Downregulation of Hepatic Cytochrome P450 in Chronic
`Renal Failure
`
`J Am Soc Nephrol 12: 326 –332, 2001
`
`FRANCOIS LEBLOND,* CARL GUÉVIN,* CHRISTIAN DEMERS,†
`ISABELLE PELLERIN,* MARIELLE GASCON-BARRÉ,† and VINCENT PICHETTE*
`*Service de Néphrologie et Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, and †Centre
`Hospitalier Universitaire de Montréal, Faculté de Médecine, Université de Montréal, Québec, Canada.
`
`Abstract. Chronic renal failure (CRF) is associated with a
`decrease in drug metabolism. The mechanism remains poorly
`understood. The present study investigated the repercussions of
`CRF on liver cytochrome P450 (CYP450). Three groups of rats
`were defined: control, control paired-fed, and CRF. Total
`CYP450 activity, protein expression of several CYP450 iso-
`forms as well as their mRNA, and the in vitro N-demethylation
`of erythromycin were assessed in liver microsomes. The reg-
`ulation of liver CYP450 by dexamethasone and phenobarbital
`was assessed in CRF rats. Compared with control and control
`paired-fed rats, creatinine clearance was reduced by 60% (P ,
`0.01) in CRF rats. Weight was reduced by 30% (P , 0.01) in
`control paired-fed and CRF rats, compared with control ani-
`mals. There was no difference in the CYP450 parameters
`between control and control paired-fed. Compared with control
`
`paired-fed rats, total CYP450 was reduced by 47% (P , 0.001)
`in CRF rats. Protein expression of CYP2C11, CYP3A1, and
`CYP3A2 were considerably reduced (.40%, P , 0.001) in
`rats with CRF. The levels of CYP1A2, CYP2C6, CYP2D, and
`CYP2E1 were the same in the three groups. Northern blot
`analysis revealed a marked downregulation in gene expression
`of CYP2C11, 3A1, and 3A2 in CRF rats. Although liver
`CYP450 was reduced in CRF, its induction by dexamethasone
`and phenobarbital was present. N-demethylation of erythromy-
`cin was decreased by 50% in CRF rats compared with control
`(P , 0.001). In conclusion, CRF in rats is associated with a
`decrease in liver cytochrome P450 activity (mainly in
`CYP2C11, CYP3A1, and 3A2), secondary to reduced gene
`expression.
`
`Reduction in renal function alters the disposition of many
`drugs mainly by decreasing the elimination of those excreted
`by the kidney (1,2). However, drug metabolism by the liver
`may also be altered in patients with chronic renal failure (CRF)
`(3). Indeed, several studies have shown that the metabolic
`clearance of various substrates is reduced in patients with CRF
`(1,3,4). The severity of the inhibition of drug metabolism is
`variable (from 17 to 85%), depending on the metabolic path-
`way involved, but drugs metabolized by hepatic cytochrome
`P450 seem particularly vulnerable (3,5). Supporting the hy-
`pothesis that CRF inhibits liver P450 is the reduction in hepatic
`P450 as well as other cytosolic enzymes in rats with experi-
`mental renal failure (6 –9).
`Rat hepatic cytochrome P450 is composed of several iso-
`forms. Those involved in drug metabolism processes include
`CYP1A2, CYP2C11, CYP2D, CYP2E1, and CYP3A1/3A2
`(10). The knowledge of which isoforms are reduced by CRF is
`critical to predict which drugs are at risk for accumulation.
`Although previous work has focused on the repercussions of
`
`CRF on total hepatic P450 content, only one has studied
`whether the reduction in P450 involves all isoforms. Uchida et
`al. (11) demonstrated that in CRF rats, protein expression of
`some liver P450 isoforms were decreased (CYP2C6, 2C11, and
`3A2) while another was increased (CYP1A1). However, the
`relation between reduced protein expression in P450 isoforms
`and their metabolic activities has not been studied. Further-
`more, the mechanism of liver P450 reduction remains poorly
`understood; it may be secondary to a decrease in synthesis or
`an increase in degradation.
`The objectives of this study were to determine the effects of
`CRF on hepatic P450 and to define the mechanisms leading to
`its downregulation. For this purpose, in control, control paired-
`fed, and CRF rats, we measured the following: (1) liver cyto-
`chrome P450 total activity; (2) the main P450 isoforms in-
`volved in drug metabolism, e.g., CYP1A1/1A2, 2C6, 2C11,
`2D1, 2E1, 3A1, and 3A2, as well as some of their specific
`metabolic activities; and (3) the mRNA encoding for these
`specific isoforms. Finally, the effect of CRF on liver cyto-
`chrome P450 was also studied in rats treated with known
`inducers of P450, dexamethasone, and phenobarbital (12).
`
`Received February 2, 2000. Accepted June 21, 2000.
`Correspondence to Dr. Vincent Pichette, Centre de Recherche Guy-Bernier,
`Hôpital Maisonneuve-Rosemont, 5415 boul. de l’Assomption, Montréal, Qué-
`bec H1T 2M4, Canada. Phone: 514-252-3489; Fax: 514-255-3026; E-mail:
`vincent.pichette@hmr.qc.ca
`1046-6673/1202-0326
`Journal of the American Society of Nephrology
`Copyright © 2001 by the American Society of Nephrology
`
`Materials and Methods
`Experimental Model
`Male Sprague-Dawley rats (Charles River, Saint-Charles, Québec,
`Canada), weighing 200 to 300 g, were housed in the Research Center
`animal care facility and maintained on Purina rat pellets (Ralston-
`Purina, St. Louis, MO) and water ad libitum. The animals were
`
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`J Am Soc Nephrol 12: 326 –332, 2001
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`Decreased Cytochrome P450 in Renal Failure
`
`327
`
`allowed an acclimatization period of at least 3 d before any experi-
`mental work was undertaken. All of the experiments were conducted
`according to the Canadian Council on Animal Care guidelines for care
`and use of laboratory animals.
`
`Experimental Protocol
`Studies were performed in three groups of 10 animals each: control,
`control paired-fed, and CRF. To evaluate further the effects of CRF on
`the regulation of liver P450, three other groups (n 5 6 in each group)
`were studied: CRF, CRF treated with dexamethasone, or CRF treated
`with phenobarbital. Dexamethasone and phenobarbital are potent in-
`ducers of liver P450 (12).
`Chronic renal failure was induced by a two-staged, five-sixths
`nephrectomy. Briefly, the rats underwent a two-thirds nephrectomy of
`the left kidney through a midline incision, and 7 d later, the right
`nephrectomy was done. Rats from both control groups underwent two
`sham laparotomies. Pentobarbital was used for anesthesia (60 mg/kg
`via intraperitoneal injection). After surgery, CRF animals were fed
`Purina rat chow and water ad libitum. Control rats were fed ad
`libitum. Control paired-fed rats were fed the same amount of rat chow
`that was ingested by the CRF rats on the previous day to assess the
`effect of CRF-induced malnutrition. Body weight was measured every
`other day for the duration of the study. At day 41 after the nephrec-
`tomy, the rats were housed in metabolic cages and urine was collected
`for 24 h to determine the clearance of creatinine. Rats were killed by
`decapitation at 42 d after nephrectomy. Blood was collected for the
`measurement of serum creatinine and urea. Enzyme induction of liver
`P450 was achieved using an intraperitoneal injection of dexametha-
`sone (100 mg/kg per d) or phenobarbital (80 mg/kg) on days 38 to 41
`(12).
`
`Preparation of Liver Microsomes
`The rat livers were immediately excised after death, and micro-
`somes were isolated by differential centrifugation (13). Samples were
`maintained at 4°C during microsome preparation. Briefly, 5 g ofliver
`was homogenized, using a Potter-Elvehjem tissue grinder (Wheaton
`Science Products, Millville, NJ), in 25 ml of 0.25 M sucrose and was
`centrifuged at 12,000 3 g. To the 12,000 3 g supernatant, 1 M CaCl2
`was added (10% vol/vol) and further centrifuged at 27,000 3 g. The
`pellet containing the microsomes was stored at 280°C in Tris 0.1 M
`(pH 7.4), glycerol 20%, ethylenediaminetetraacetate (EDTA) 10 mM
`up to analysis.
`
`Determination of Total Cytochrome P450 Activity
`Microsomal protein content was determined by the method of
`Lowry et al. (14), using bovine serum albumin as standard protein.
`Total cytochrome P450 activity was measured from the difference
`spectrum of the reduced protein according to a previously published
`method (15).
`
`Western Blot Analysis
`The major cytochrome P450 isoforms implicated in the metabolism
`of drugs were assessed by Western blot analysis: CYP1A2, CYP2C6,
`CYP2C11, CYP2D, CYP2E1, CYP3A1, and CYP3A2. Forty mg of
`protein was electrophoresed in a 7.5% polyacrylamide gel containing
`0.1% sodium dodecyl sulfate (SDS), and separated proteins were
`electrophoretically transferred on nitrocellulose (16,17). Immunoblots
`for respective isoforms were performed in 5% low-fat milk in phos-
`phate-buffered saline and washed with 0.1% Tween 20 in phosphate-
`buffered saline. CYP1A2 was detected using a polyclonal goat anti-rat
`1A2 (Gentest Corporation, Woburn, MA). CYP2C6 and CYP2C11
`
`were detected using a goat anti-rat 2C6 and 2C11 (Gentest), respec-
`tively. CYP2D was detected using a rabbit anti-human 2D (Oxford
`Biochemical Research Inc., Oxford, MI). CYP2E1 and CYP3A1 were
`detected using a monoclonal mouse anti-rat 2E1 and 3A1 (Oxford),
`respectively. CYP3A2 was detected using a goat anti-rat 3A2 (Gen-
`test). Immune complexes were revealed by secondary antibody (swine
`anti-goat IgG and goat anti-rabbit IgG [Biosource International, Cam-
`marillo, CA], as well as goat anti-mouse IgG [Sigma Chemicals, St.
`Louis, MO]) coupled to peroxidase and the Luminol derivative of
`Lumi-Light Western blotting substrate (Roche Diagnostics, Laval,
`Québec, Canada). Immune reaction intensity was determined by com-
`puter-assisted densitometry on exposed Biomax MR film (Scientific
`Imaging Systems, Eastman Kodak Co., Rochester, NY).
`
`mRNA Analysis
`At the time of death, biopsies of liver were rinsed in ice-cold saline
`and flash-frozen in liquid nitrogen. Samples were kept at 280°C until
`RNA extraction. The RNA encoding for CYP1A2, CYP2C11,
`CYP3A1, and CYP3A2 was evaluated by Northern blot analysis.
`Total RNA was extracted from frozen tissue by the RNeasy kit
`(Qiagen, Mississauga, Ontario, Canada). RNA concentrations were
`determined by measuring absorbance at a wavelength of 260 nm.
`Total RNA samples were denatured by heating at 65°C in buffer
`containing 42% deionized formamide, 30 mM 4-morpholinepropane-
`sulfonic acid, and 8.5% formaldehyde. RNAs (30 mg total RNA) were
`separated by electrophoresis in 1% agarose-1.7% formaldehyde gel
`submerged in buffer (pH 7.2), containing 20 mM 4-morpholinepro-
`panesulfonic acid, 8 mM sodium acetate, and 1 mM EDTA. Separate
`RNA were transferred to nylon membranes (Qiabrane, Qiagen), using
`the standard capillary technique with 10 3 SSC (1.5 M NaCl and 0.15
`M sodium citrate, pH 7.0) and fixed under ultraviolet lamp at 0.6
`J/cm2. Prehybridizations were performed at 52°C in buffer composed
`of 0.5 M NaPO4 (pH 7.2), 7% SDS, 1% bovine serum albumin, 1%
`dextran sulfate, 1 mM EDTA, and 250 mg/ml denatured herring sperm
`DNA (Roche Diagnostics). The blots were hybridized with oligonu-
`cleotide probes specific for each P450 mRNA: for CYP1A2, a syn-
`thetic 20-mer oligonucleotide complementary to bases 1580 to 1599
`of the P450 1A2 cDNA sequence (18); for CYP2C11, a 30-mer
`oligonucleotide corresponding to the complement of nucleotides 945
`to 974 of the coding sequences of P450 2C11 (19); for CYP3A1, a
`synthetic 32-mer oligonucleotide complementary to bases 1593 to
`1624 of the P450PCN1 nucleotide sequence (20); for CYP3A2, a
`synthetic 24-mer oligonucleotide complementary to bases 1652 to
`1675 of the 6b-A nucleotide sequence (21); for 18S ribosomal RNA,
`a 1.5-kb human cDNA insert from EcoRI site of the pBluescript
`SK-vector ATCC no.77242 (MBI Fermentas). Oligonucleotides were
`prepared by the Sheldon Biotechnology Center (McGill University,
`Montreal, Québec, Canada). They were end labeled with [g-32P]ATP
`(3000 Ci/mmol), using T4 polynucleotide kinase (BRL, Burlington,
`Ontario, Canada). The 18S cDNA probe was
`labeled using
`[a-32P]dCTP (3000 Ci/mmol) and Klenow according to the random
`oligo-priming method. Hybridization was then performed at 52°C for
`24 h in prehybridization buffer to which were added the labeled
`probes. The membranes were then washed in 0.2 M NaPO4 (pH 7.2),
`1% SDS, 1 mM EDTA at room temperature and at 52°C. Washed
`membranes were exposed to autoradiography film (Biomax MS;
`Kodak) with Biomax TranScreen-HE intensifying screen (Kodak) at
`280°C for 3 to 10 d. Hybridization signals were quantified by
`computer-assisted densitometer. mRNA levels were expressed as ar-
`bitrary densitometric units and standardized by comparison with hy-
`bridization results obtained with 18S ribosomal RNA probe.
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`328
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`Journal of the American Society of Nephrology
`
`J Am Soc Nephrol 12: 326 –332, 2001
`
`In Vitro Metabolism of Erythromycin
`To evaluate the metabolic activity of CYP3A2 in liver microsomes,
`erythromycin N-demethylation was determined as described by Wang
`et al. (22). Erythromycin (250 mM) (Sigma Chemicals) was incubated
`with 2.0 mg of rat liver microsomes (either from control paired-fed or
`CRF) at 37°C for 15 min in the presence of an NADPH-generating
`system consisting of the following: 10 mM glucose 6-phosphate, 1
`mM NADP, and 0.35 units glucose-6-phosphate dehydrogenase (Sig-
`ma Chemicals) in a total volume of 0.5 ml. Reactions were quenched
`with 0.05 ml of 25% ZnSO4 and 0.05 ml of 0.3N Ba(OH)2. The
`samples were then centrifuged at 14,000 3 g for 10 min, and 0.35 ml
`of the supernatant was transferred and mixed with 0.15 ml of Nash
`reagent (23). The mixture was incubated at 56°C for 30 min, and
`samples were analyzed by spectrophotometry (absorbance 405 nm) to
`determine the formation of formaldehyde (23).
`
`Other Assays
`Blood and urine chemistries were determined with a Hitachi 717
`autoanalyser
`(Boehringer Mannheim Canada, Laval, Québec,
`Canada).
`
`Statistical Analyses
`The results are expressed as mean 6 SEM. Differences between
`groups were assessed by using an unpaired t test or an ANOVA test.
`Significant ANOVA was followed by Fisher least significant differ-
`ence multiple comparisons procedure. The threshold of significance
`was P , 0.05
`
`Results
`Biochemical Parameters and Body Weight in Control,
`Control Paired-Fed, and CRF Rats
`Table 1 presents the biochemicals and body weight of the
`three groups of animals studied. Compared with control and
`control paired-fed animals, CRF rats had higher levels of
`plasma creatinine and urea and lower values of creatinine
`clearance (reduced by 60%; P , 0.001). Body weight in
`control paired-fed and CRF rats was reduced by 30% compared
`with control animals. However, there was no difference in
`body weight between control paired-fed and CRF rats.
`
`Liver Total Cytochrome P450 Activity in Control,
`Control Paired-Fed, and CRF Rats
`No difference was observed in total cytochrome P450 ac-
`tivity between control and control paired rats (0.61 6 0.01 and
`0.59 6 0.04 nmol/mg protein; Table 1). Thus, malnutrition as
`
`produced by CRF has no effect on liver P450. In CRF rats, total
`cytochrome P450 activity was significantly reduced by 47%
`compared with both control groups (Table 1). The P450 activ-
`ity was negatively correlated with creatinine clearance (r 5
`0.68, P , 0.001). Similar correlations were found between
`P450 activity and blood urea and creatinine.
`
`Protein Expression of Liver Cytochrome P450 Isoforms
`in Control, Control Paired-Fed, and CRF Rats
`No differences in the different isoforms between control and
`control paired-fed rats were observed (data not shown). The
`levels of CYP2C11, 3A1, and 3A2 in CRF rats were reduced
`by 40, 75, and 65%, respectively, in CRF rats compared with
`control paired-fed animals (P , 0.001; Figure 1). Conversely,
`the levels of CYP1A2, 2C6, 2D1, and 2E1 were not modified
`in CRF rats compared with control rats.
`
`mRNA Encoding Liver Cytochrome P450 Isoforms in
`Control, Control Paired-Fed, and CRF Rats
`To determine whether liver cytochrome P450 isoforms in
`CRF were downregulated secondary to a decrease in their
`synthesis or an increase in their degradation, we evaluated
`mRNA encoding the different isoforms by Northern blot anal-
`ysis. Again, there was no difference in the level of mRNA
`between control and control paired-fed rats (data not shown).
`However,
`a
`significant decrease
`in mRNA encoding
`CYP2C11, 3A1, and 3A2 isoforms was observed in CRF
`compared with control paired-fed animals (Figure 2). Thus, the
`decrease in protein expression of the different isoforms of P450
`observed in CRF is secondary to reduced gene expression.
`
`In Vitro Metabolism of Erythromycin in Control,
`Control Paired-Fed, and CRF Rats
`To determine the repercussion of cytochrome P450 reduction in
`CRF on the metabolism of drugs, we assessed the in vitro N-
`demethylation of erythromycin in liver. This enzymatic reaction is
`mediated primarily by the CYP3A family. No differences be-
`tween control and control paired-fed rats were observed (data not
`shown). The N-demethylation of erythromycin was decreased by
`more than 50% in rats with CRF, compared with control paired-
`fed animals (P , 0.001; Figure 3).
`
`Table 1. Characteristics of the control, control paired-fed and CRF ratsa
`
`Control
`
`Control Paired-Fed
`
`CRF
`
`Body weight (g)
`Serum creatinine (mmol/L)
`Creatinine clearance (ml/100 g of body weight/min)
`Serum urea (mmol/L)
`Liver total cytochrome P450 activity (nmol/mg of proteins)
`
`459 6 3.7
`52 6 1
`406 6 18
`4.8 6 0.2
`0.61 6 0.01
`
`318 6 9.4b
`56 6 2
`390 6 17
`5.9 6 0.5
`0.59 6 0.04
`
`327 6 8.5b
`141 6 9b,c
`178 6 25b,c
`24.5 6 5.8b,c
`0.34 6 0.04b,c
`
`a CRF, chronic renal failure. Data are the mean 6 SEM. Measurements were made 41 d after the first surgery.
`b P , 0.01 compared with control rats.
`c P , 0.01 compared with control paired-fed animals.
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`J Am Soc Nephrol 12: 326 –332, 2001
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`Decreased Cytochrome P450 in Renal Failure
`
`329
`
`Figure 1. Protein expression of liver cytochrome P450 isoforms in control paired-fed (e) and chronic renal failure (CRF) rats (f). Protein bands
`are expressed in densitometry units (%). The densitometry units of control paired-fed rats were arbitrarily defined as 100%. Data are the mean
`6 SEM of 10 rats in each group. *, P , 0.001 as compared with control paired-fed rats. Representative blots are also shown.
`
`Figure 2. mRNA encoding liver cytochrome P450 isoforms (CYP1A2, CYP2C11, CYP3A1, and 3A2) in control paired-fed (e) and CRF rats
`(f). mRNA bands are expressed in standardized densitometry units (%). The densitometry values for cytochrome P450 isoforms were
`standardized by dividing these values by the values for 18S ribosomal RNA. The standardized densitometry units of control paired-fed rats were
`arbitrarily defined as 100%. Data are the mean 6 SEM of six rats in each group. *, P , 0.01 as compared with control paired-fed rats.
`Representative blots are also shown.
`
`Induction of Cytochrome P450 in Rats with CRF by
`Dexamethasone or Phenobarbital
`Although our results showed that the decrease in liver cy-
`tochrome P450 in CRF is secondary to reduced gene expres-
`sion, we were also interested to know whether P450 was still
`inducible, despite its inhibition by CRF. We studied the effect
`of dexamethasone and phenobarbital (which are potent induc-
`ers of the CYP3A family) in CRF rats on CYP3A1 and 3A2
`protein expressions as well as on their mRNA levels (Figure 4).
`In CRF rats, CYP3A1 and 3A2 were greatly enhanced by
`dexamethasone and phenobarbital. This was secondary to an
`upregulation of mRNA encoding these proteins. We further
`studied the effect of dexamethasone on N-demethylation of
`
`erythromycin. In CRF rats, N-demethylation of erythromycin
`was also increased by dexamethasone (0.28 6 0.02 versus 1.38
`6 0.07 nmol/mg of protein/min, P , 0.001).
`
`Discussion
`This study demonstrates that in the rat, CRF induces a
`marked decrease in liver total cytochrome P450 activity sec-
`ondary to reduced protein expression of selective cytochrome
`P450 isoforms, namely CYP2C11, 3A1, and 3A2. The mech-
`anism underlying this downregulation is a reduction in the
`mRNA levels encoding these proteins. The repercussions on
`the metabolism of drugs by the liver are important in that we
`observed a 50% reduction of erythromycin biotransformation
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`Journal of the American Society of Nephrology
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`J Am Soc Nephrol 12: 326 –332, 2001
`
`Our results demonstrate that there is an association between
`lower levels of mRNA and protein for some isoforms of
`cytochrome P450, namely CYP2C11, 3A1, and 3A2 (Figure
`2). This suggests that there is reduced gene expression. The
`mechanisms responsible for the diminished liver CYP2C11,
`3A1, and 3A2 gene expression in CRF are not known. Caloric
`restriction, as seen in CRF rats, downregulates hepatic genes of
`drug metabolizing enzymes in the mouse and in the rat (25,26).
`However, in our control paired-fed rats, we did not observe any
`modification in cytochrome P450 levels despite a similar
`weight loss as in CRF rats. Further studies will require evalu-
`ation of uremia on the function of the CYP450 promoters.
`A number of studies indicate that animals with CRF display
`impaired protein synthesis, by reduced gene expression, in the
`liver as well as in the skeletal muscle (27,28). For instance, the
`mRNA of hepatic lipase and insulin-like growth factor 1 re-
`ceptor are decreased in hepatocytes and skeletal muscle, re-
`spectively, of rats with CRF (27,28). CRF is associated with
`sustained elevations in calcium in many cell types, including
`the hepatocytes as well as the skeletal muscle, and this high
`intracellular calcium seems to be a major factor underlying cell
`dysfunction and reduced protein synthesis in CRF (29,30).
`However, the effects of intracellular calcium on the regulation
`of P450 remain poorly defined (31,32). Whether increased
`intracellular calcium is implicated in the downregulation of
`liver P450 in CRF remains to be studied.
`In the present study, downregulation of cytochrome P450 in
`CRF was overcome by dexamethasone and also by phenobar-
`bital, which are potent inducers of CYP3A isoforms (Figure 4).
`This result suggests that although liver cytochrome P450 is
`decreased in CRF, its dexamethasone and phenobarbital regu-
`lation is still present. The clinical significance of this finding
`remains to be defined. However, one can anticipate that in
`CRF, liver cytochrome P450 could be modulated by known
`P450 inducers, e.g., steroids, phenobarbital.
`Several investigators have demonstrated that in patients with
`renal failure, there is also a decrease in the metabolic clearance
`of many drugs (ranging from 17 to 85%) (1,3,4). The vast
`majority of these drugs are metabolized by the liver through the
`cytochrome P450 pathway. Unfortunately, we are not aware of
`human studies demonstrating a reduction in liver cytochrome
`P450 isoforms in CRF. However, several methods have been
`described to assess in vivo the CYP activity in humans, and the
`most widely used is the administration of probe drugs that are
`selectively metabolized by a specific CYP isoform. Kevorkian
`et al. (33) reported that assessment of CYP2D6 activity by the
`use of dextromethorphan or sparteine was possible in patients
`with CRF . The results of their study revealed that there was a
`decrease in the metabolic clearance of sparteine suggesting a
`decrease in CYP2D6 activity in CRF patients. There seems to
`be a correlation between the decrease in the metabolism of
`drugs and the severity of renal failure in humans (34). Inter-
`estingly, in the rat, we found a significant correlation between
`the decrease in renal function and the reduction in liver cyto-
`chrome P450 and also with the reduction in the N-demethyl-
`ation of erythromycin. These results suggest
`that as CRF
`
`Figure 3. In vitro metabolism of erythromycin (N-demethylation) in
`control paired-fed (e) and CRF rats (f). Incubations were performed
`with 2 mg of rat liver microsome at 37°C for 15 min. Data are the
`mean 6 SEM of six rats in each group. *, P , 0.001 as compared with
`control paired-fed rats.
`
`mediated by the CYP3A family. Although liver cytochrome
`P450 is decreased in CRF, its dexamethasone and phenobar-
`bital regulation was still present as shown by the induction of
`CYP3A by dexamethasone or phenobarbital.
`Renal failure has been generally thought to decrease only the
`renal clearance of drugs (24). However, several studies have
`demonstrated that animals with CRF also present decreased
`hepatic drug metabolism (5). Because the P450 is the major
`enzymatic system involved in drug metabolism, most studies
`have focused on liver P450. The results of these studies show
`that in CRF rats there is a 18.6 to 42.8% decrease in liver total
`P450 (6 –9,11). Furthermore, important reductions in enzy-
`matic reactions normally carried by the liver P450 have been
`reported: N-demethylation of aminopyrine and ethylmorphine,
`O-demethylation of codeine, and hydroxylation of aniline (5).
`In the present study, we found a 47% reduction in total P450
`activity as well as a significant reduction in the N-demethyl-
`ation of erythromycin.
`Few studies have focused on the specific P450 isoforms
`reduced in CRF (11). Knowledge of which isoform is reduced
`by CRF is critical to predict which drugs are at risk for
`accumulation when used in CRF. Recently, Uchida et al. (11)
`reported a reduction in the levels of hepatic CYP2C6,
`CYP2C11, and CYP3A2 and a slight increase in CYP1A2 in
`rats with CRF. Our results demonstrate that only CYP2C11,
`CYP3A1, and CYP3A2 are significantly reduced, while no
`isoform induction was noted. Furthermore, the level of reduc-
`tion was far more important in the present study. This could
`reflect a more pronounced degree of uremia obtained in our
`rats (50% increase in plasma creatinine versus 65% in the
`present study) and also a longer period of uremia (21 d versus
`42 d in the present study). Interestingly, CYP3A1 and 3A2 in
`the rat correspond to CYP3A4 in humans. Because this isoform
`is responsible for the metabolism of several drugs commonly
`used in CRF patients, patients with CRF could be at risk for
`drug accumulation and toxicity.
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`J Am Soc Nephrol 12: 326 –332, 2001
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`Decreased Cytochrome P450 in Renal Failure
`
`331
`
`Figure 4. Changes in liver microsome levels of CYP3A2 protein expression and specific mRNA in response to treatment with dexamethasone
`(100 mg/kg intraperitoneally daily during 4 d) (f) or with phenobarbital (80 mg/kg intraperitoneally daily during 4 d; &U25A8;). Data are the
`mean 6 SEM of six rats in each group. *, P , 0.001 as compared with CRF without inducer treatment (e). Representative blots are also shown.
`
`worsened, patients were at risk of drug accumulation and
`toxicity, secondary to reduction in their metabolism.
`In conclusion, CRF is associated with a decrease in liver
`cytochrome P450 isoforms
`in rats
`(mainly CYP2C11,
`CYP3A1, and 3A2), secondary to reduced mRNA levels. Drug
`metabolism activity, assessed by the N-demethylation of eryth-
`romycin, is also greatly depressed in CRF rats. Liver cyto-
`chrome P450 downregulation is correlated with the degree of
`renal failure. This decrease could explain the reduction in drug
`metabolism observed in CRF patients because these isoforms
`(especially CYP3A1 and 3A2) correspond to CYP3A4 in hu-
`mans, which is responsible for the metabolism of several drugs
`commonly used in CRF patients.
`
`Acknowledgments
`This work was supported by the Kidney Foundation of Canada and
`Fonds de la Recherche en Santé du Québec. Part of this work has been
`presented at the 31st and 32nd annual meetings of the American
`Society of Nephrology in Philadelphia and Miami, respectively. Vin-
`cent Pichette is a scholar of the Kidney Foundation of Canada.
`
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