Downregulation of Hepatic Acetylation of Drugs in Chronic Renal Failure
Emilie Simard*,,
Judith Naud*,,
Josée Michaud*,,
Francois A. Leblond*,
Alain Bonnardeaux*,
Chantal Guillemette,
Edith Sim and
Vincent Pichette*,
* Service de néphrologie et Centre de recherche de l'Hôpital Maisonneuve-Rosemont and Département de pharmacologie, Université de Montréal, Montréal, and Faculté de pharmacie, Université Laval, Laval, Québec, Canada; and Department of Pharmacology, University of Oxford, Oxford, United Kingdom
Correspondence: Dr. Vincent Pichette, Centre de recherche, Hôpital Maisonneuve-Rosemont, 5415 boulevard de l'Assomption, Montréal, Québec, Canada, H1T 2M4. Phone: 514-252-3489; Fax: 514-255-3026; E-mail: vpichette.hmr{at}ssss.gouv.qc.ca
Received for publication September 4, 2007.
Accepted for publication February 2, 2008.
Drug metabolism can be affected by chronic renal failure (CRF).Although it is known that several drugs that are known to beacetylated accumulate in CRF, the effect of CRF on N-acetyltransferase(NAT), the enzyme responsible for this acetylation, is unknown.Herein is reported that protein and gene expression of bothNat isoforms in the liver was reduced by >30% and Nat2 activitywas reduced by 50% in rats with CRF compared with control rats.Incubation of hepatocytes with serum from rats with CRF suggestedthat a circulating factor is responsible for the decrease inprotein and gene expression. For testing the hypothesis thatparathyroid hormone may be this factor, CRF was induced in parathyroidectomizedrats; downregulation of Nat expression and activity was notobserved in these rats. Furthermore, addition of parathyroidhormone to cultured hepatocytes induced a decrease in Nat2 proteinand gene expression. In conclusion, liver acetylation of drugsin a rat model of CRF is reduced by a downregulation of Nat1and Nat2 isoforms, secondary to decreased gene expression. Parathyroidhormone seems to be an important mediator of this phenomenon.
Reduction in renal function alters the disposition of many drugsmainly by decreasing the elimination of those excreted by thekidney1,2; however, drug metabolism by the liver may also bealtered in patients with chronic renal failure (CRF).3 Indeed,several studies have shown that the metabolic clearance of varioussubstrates is reduced in patients with CRF.1–10 Liverenzymes implicated in drug metabolism are classified in phaseI (mainly oxidative reaction mediated by cytochrome P450) andphase II (conjugation reactions). Up to now, the studies onthe repercussions of CRF on drug metabolism have been primarilyfocused on cytochrome P450. Indeed, several studies have demonstratedthat CRF in rats was associated with a reduction in proteinand gene expression as well as a significant decrease in theactivity of several liver cytochrome P450 isoforms.11–16Moreover, we recently reported that this decrease was secondaryto uremic circulating factors, particularly parathyroid hormone(PTH).17
Phase II enzymes are also critical for the metabolism of drugs.18,19These enzymes are responsible for the conjugation of drugs,and glucuronidation mediated by uridine diphosphate-glucuronosyltransferase (UGT) and acetylation mediated by N-acetyltransferases(NAT) are among the most important reactions.19 Interestingly,several human studies have reported a reduction in the livermetabolism of drugs that are cleared by glucuronidation or acetylationin patients with CRF, suggesting that CRF could impede thesemetabolic pathways.6–9 For instance, zidovudine and morphine(two glucuronidated molecules) have a significantly reducedmetabolic clearance in CRF.20,21 Moreover, several drugs extensivelyacetylated (e.g., isoniazid, procainamide) also accumulate inpatients with decreased renal function,2,22–24 suggestinga reduction in hepatic acetylation. Recently, Yu et al.25 showedthat liver UGT was unchanged in a CRF rat model; however, nostudy has been done on the effect of CRF on hepatic acetylationin rat.
Human NAT are composed of two isoforms (NAT1 and NAT2), andboth are involved in drug metabolism processes, although humanNAT1 is also thought to have an endogenous role.26 These enzymesare expressed in the liver; they catalyze acetyl group transferfrom acetyl-CoA to an aromatic or heterocyclic amine, hydrazine,hydrazide, or N-hydroxylamine acceptor substrate. NAT metabolizeseveral drugs, such as sulfonamides and isoniazid.26 In therat, similar to human, there are two isoforms (Nat1 and Nat2),although a third isoform (Nat3) with less metabolic activitywas recently characterized.27
The objectives of this study were to determine the effects ofCRF on hepatic NAT and to define the mechanisms leading to theirdownregulation. For this purpose, we measured in control andCRF rats (1) liver Nat1 and 2 protein and gene expression and(2) specific metabolic activity of Nat2. We also evaluated theeffects of uremic serum on Nat1 and Nat2 expression by incubatingnormal rat hepatocytes with control or CRF sera. Finally, theimplication of PTH (as a potential uremic factor) in the downregulationof rat Nat genes was also evaluated in vivo in parathyroidectomizedrats and in vitro in rat hepatocytes incubated with PTH.
Biochemical Parameters and Body Weight in Control and CRF Rats Table 1 presents the body weight and the biochemical parametersof the animals studied. As shown, body weights were similarin all groups. CRF rats had higher levels of plasma creatinineand a lower creatinine clearance reduced by 79% (P < 0.001)compared with control rats.
Nat Isoforms in the Liver
The levels of Nat1 and Nat2 in CRF rat (n = 10) liver were reducedby 33 and 50%, respectively, compared with control animals (P< 0.001; Figure 1). There was a significant negative correlationof Nat protein levels and creatinine clearance (r = 0.76, P< 0.001). To evaluate whether the downregulation of Nat inthe liver of CRF rat was secondary to a decrease in the proteinssynthesis, we evaluated mRNA encoding the different isoformsby multiplex quantitative PCR. This reveals a significant decreasein mRNA encoding Nat1 and Nat2 in CRF animals (n = 19) comparedwith controls (n = 16; Figure 2). Thus, the decrease in proteinlevels of the different Nat isoforms observed in CRF is secondaryto reduced gene expression.
Figure 1. Protein expression of liver Nat1 and 2 in control (CTL; ) and CRF () rats. Control results were defined as 100%. Data are means ± SEM. Representative blots are shown in insert. *P < 0.001 versus CTL rats.
Figure 2. Expression of liver Nat1 and 2 mRNA in CTL () and CRF () rats. Control results were defined as 100%. Data are means ± SEM, **P < 0.01 versus CTL rats.
In Vitro Metabolism of p-Aminobenzoic Acid
To determine the repercussion of CRF on the metabolism of drugsby Nat, we assessed the in vitro N-acetylation of p-aminobenzoicacid (PABA; Sigma-Aldrich, Oakville, ON, Canada) in liver cytosols.This enzymatic reaction was mediated primarily by the rat Nat2isoform. The N-acetylation of PABA was decreased by 50% in ratswith CRF (n = 8), compared with control animals (P < 0.001;n = 6; Figure 3).
Figure 3. Nat2 metabolizing activity as measured by using PABA as specific substrate in CTL () and CRF () rats liver. Results expressed by nmol of N-acetyl-PABA (NAPABA) produced per mg of protein per minute. Data are means ± SEM. *P < 0.001 versus CTL rats.
Effects of Uremic Serum on Nat Protein and mRNA Levels
The objective of this experiment was to determine the effectsof CRF serum on liver Nat. After 48 h of incubation of normalhepatocytes with serum (10%) from rats with CRF (n = 15), theamounts of Nat1 and 2 proteins were decreased by 40 and 57%(P < 0.005), respectively, compared with hepatocytes incubatedwith serum from control animals (n = 12; Figure 4A). The mRNAlevels of Nat1 and Nat2 were both decreased by 30% (P < 0.005;Figure 4B). Thus, uremic serum contains mediators that downregulateNat expression in normal hepatocytes.
Figure 4. (A and B) Protein (A) and mRNA (B) expression of Nat1 and Nat2 in 48-h cultured hepatocytes incubated with CTL () and CRF () rat serum (10%). Expression in hepatocytes incubated with CTL serum was defined as 100%. Representative blots are shown in insert. Data are means ± SEM. P < 0.005 versus CTL rats; *P < 0.001 versus CTL rats.
Physical and Biochemical Characteristics of Rats with Parathyroidectomy
For clarification of the role of PTH in the downregulation ofNat genes, some CRF (n = 6) and control (n = 6) rats were surgicallytreated to remove parathyroid glands. Table 2 presents the bodyweight and the biochemical characteristics of the animals usedin this experiment. Once again, no difference was observed betweengroups for body weights, whereas clearance and creatinine weresignificantly reduced in both CRF groups. There was a 40-foldincrease in PTH in CRF rats, confirming the severity of secondaryhyperparathyroidism in these animals. PTH was undetectable inrats with parathyroidectomy (PTX).
Table 2. Physiologic characteristics of rats with PTXa
Role of PTH in the In Vivo Downregulation of Rat Nat1 and Nat2 Induced by CRF
To determine whether PTH was implicated in the in vivo decreaseof Nat by CRF, we evaluated the effects of PTX in CRF rats onNat1 and 2 protein levels. As shown in Figure 5, preventingthe development of secondary hyperparathyroidism by PTX preventsthe negative effect of CRF on Nat2 protein and the Nat2 gene.We also observed a significant difference in Nat2 metabolicactivity in CRF rats with PTX compared with CRF rats (Figure 5).Similar results were obtained for Nat1 (Figure 6).
Figure 5. Expression of Nat2 protein (), mRNA () and activity (
) in CTL and CRF rat liver with or without PTX. CTL rats were defined as 100%. Data are means ± SEM. Representative blots are shown in insert. P < 0.05 versus control rats; P < 0.05 versus CRF group.
Figure 6. Expression of Nat1 protein () and mRNA () in CTL and CRF rat liver with or without PTX. CTL rats were defined as 100%. Data are means ± SEM. Representative blots are shown in insert. P < 0.05 versus CTL rats.
In Vitro Effect of PTH on Nat2 in Cultured Hepatocytes
To confirm the implication of PTH in the downregulation of Nat2in CRF, we incubated normal hepatocytes during 48 h with 10–9M synthetic 1-34 rat PTH (n = 12). As shown on Table 2, thisconcentration of PTH is similar to the one observed in vivoin our CRF rats. The effects of PTH on the Nat2 protein andmRNA levels are shown on Figure 7. When hepatocytes where incubatedwith PTH, Nat2 protein and Nat2 mRNA levels were decreased by80% (P < 0.01) and 25% (P < 0.01), respectively. Therewas no effect of PTH on other control hepatocyte proteins (glyceraldehyde3-phosphate dehydrogenase, aspartate aminotransferase, or β-actin;data not shown).
Figure 7. Protein () and mRNA () expression of Nat2 in hepatocytes incubated for 48 h with 10–9 M PTH in 10% calf serum with or without 30 µM andrographolide (andro). Results of samples incubated with calf serum without PTH and andrographolide were defined as 100%. Data are means ± SEM. Representative blots are shown in insert. P < 0.05 versus CTL group; #P < 0.05 versus PTH group.
Although PTH could act via several signaling pathways, we testedthe hypothesis that the downregulation of Nat2 was through activationof NF-B, as previously shown for CYP450.17 Addition of 30 µMandrographolide to hepatocytes cultured in the presence of 10–9M PTH (n = 6) prevented the decrease in Nat2 gene expressioninduced by PTH with a subsequent effect on Nat2 protein level(Figure 7).
This study demonstrates that in the rat, CRF induces a markeddecrease in liver protein levels of Nat1 and Nat2 isoforms,secondary to a reduction in the levels of mRNA encoding theseproteins. The repercussions on the metabolism of drugs by theliver are important, because we observed a 50% reduction ofPABA metabolism mediated by Nat2 isoform. The mechanism underlyingthe downregulation of Nat genes is the presence of endogenousinhibitors in the uremic blood, PTH being one of the major inhibitors.
Enzymatic reactions leading to drug biotransformation are classifiedin phase I and phase II reactions. Phase I reactions usuallyconvert parent drugs to a more polar metabolite and are mediatedby cytochrome P450. Parent drugs or their phase I metabolitescould also undergo conjugation reactions with an endogenoussubstrate, such as glucuronic acid, acetic acid, or sulfuricacid, to yield drug conjugates. The two major phase II reactionsimplicated in drug metabolism are glucuronidation, mediatedby UGT, and acetylation, mediated by Nat.
Renal failure has been generally thought mainly to decreasethe renal clearance of drugs1,2; however, many studies havedemonstrated that humans with CRF also present decreased hepaticdrug metabolism.1–10 Because the cytochrome P450 is themajor enzymatic system involved in drug biotransformation, moststudies have focused on liver P450. The results of these studiesshowed that in CRF rats, liver total P450, several P450 isoforms,11–16,28and enzymatic reactions normally carried by the liver P450 aresignificantly decreased.10 We also showed that both patients’and rats’ uremic serum contain mediators that downregulateliver P450.29,30
Many drugs commonly used in clinical practice are metabolizedby phase II enzymes. Several pharmacokinetics studies have revealeda decrease in glucuronidation or acetylation of drugs in patientswith CRF, suggesting that not only P450 could be altered inCRF but also phase II enzymes. For instance, the metabolismof zidovudine and metoclopramide, which is primarily mediatedby glucuronidation, is reduced in patients with renal failure.20,31,32Acetylation also seems to be reduced in patients with CRF; severalreports have demonstrated that the metabolic clearance of procainamideis reduced by approximately 60% in renal failure.2,23,24 Moreover,a decrease in the acetylation of isoniazid has been reportedin patients with CRF.22
The mechanism leading to a decrease in phase II enzymatic reactionsin CRF is unknown. Although many animal studies have been doneon P450, very few have been performed on the effects of CRFon phase II enzymes. Paterson and Cohn14 studied the uridinediphosphate (UDP)-glucuronyl transferase activity in microsomesfrom control rats and rats with CRF and reported a 19% reductionin UDP-glucuronyl transferase activity, although this differencewas NS (P = 0.06). More recently, Yu et al.25 did not confirmthese results: They found no significant difference in liverUGT in CRF rats compared with control rats, although the numberof animals was small (n = 4 per group) and the degree of renalfailure was moderate. Taburet et al.,33 however, reported thatincubation of microsomes, prepared from healthy human livers,with serum of patients with CRF was associated with a decreasein the metabolism of zidovudine, reflecting an inhibition ofUDP-glucuronosyltransferase and suggesting that uremic mediatorsare responsible for the reduction in drug metabolism.
Our study is the first to evaluate the effects of CRF on acetylationof drugs. Our results demonstrate a significant decrease inboth Nat1 and Nat2 with a major impact on their in vitro activity.Interestingly, we found a strong correlation between creatinineclearance and protein expression, suggesting that as renal functiondecreases, downregulation of Nat is more important. As shownfor CYP450, the mechanism of protein downregulation is secondaryto a reduced gene expression. These results could explain whythe clearance of drugs cleared by acetylation is reduced inpatients with CRF.
Another hypothesis to explain decreased drug metabolism in CRFhave been raised by Sun et al.,34,35 who demonstrated that hepaticuptake of erythromycin (a CYP450 substrate) by hepatocytes incubatedwith uremic toxins was decreased. Because uptake by hepatictransporters (e.g., organic anion transporter protein) is thefirst step in the elimination of several drugs, making it availablefor metabolizing enzymes, a decrease in organic anion transporterprotein as shown in hepatocytes incubated with uremic toxinsand more recently in CRF rats could probably explain in partthe decrease in drug metabolism induced by CRF.34–36 Inthis study, we found a decrease in Nat activity using cytosolicpreparations that only reflect the metabolic activity and notthe uptake or the efflux of drugs that could also be modulatedin CRF.
In this study, we tested the hypothesis that uremic mediatorswere implicated in the decrease of NAT in CRF. The results clearlydemonstrate that uremic serum contains mediators that are ableto downregulate hepatic NAT. Interestingly, the results obtainedwith uremic serum also closely mimic what we showed in vivoin rats with CRF (Figures 1 and 4). Our results also demonstratethat there is an association between lower levels of Nat mRNAand the corresponding protein (Figure 4) in hepatocytes incubatedwith serum from rats with CRF. This suggests that gene expressionis reduced by mediators present in uremic serum. Although themechanisms responsible for the diminished hepatic gene expressionin CRF are not known, this study suggests that uremic mediator(s)could affect NAT promoters.
CRF is often associated with secondary hyperparathyroidism.High levels of PTH have been linked to impaired protein synthesis,by reduced gene expression, in the liver, the skeletal muscle,and the cardiomyocytes.37–39 More recently, we also showedthat PTH was an important factor implicated in the decreasedof liver P450 in CRF.17 In this study, we tested the hypothesisthat PTH could also be implicated in the decrease in liver acetylation.First, we evaluated whether PTX could prevent the downregulationof Nat genes. As shown in Figures 5 and 6, CRF rats with PTXhave essentially the same level and metabolic activity of Natas control rats, demonstrating that PTH is a major in vivo mediatorof CRF-induced downregulation of Nat2. Moreover, our resultsdemonstrate that PTH could decrease Nat2 gene expression andcause a corresponding reduction in Nat2 protein in normal hepatocytes(Figure 7).
Our results emphasize the deleterious effects of PTH on proteinsynthesis by the liver. The signaling pathway through whichthis occur remains poorly described. PTH could act by an increasein cAMP or intracellular calcium.40,41 More recently, we suggestedthat PTH could also act by activation of NF-B to downregulateliver CYP450.17 In this study, we also demonstrated that inhibitingthe activation of NF-B prevents the decrease of Nat activityinduced by PTH in culture hepatocytes. Further studies are neededto elucidate the effect of PTH on NAT and to investigate furtherthe possible role of transcription factors including NF-B.
In conclusion, this study demonstrated that CRF induces a decreasein rat Nat1 and Nat2 protein secondary to a decrease in geneexpression. We also found a major reduction of Nat2 activity,which could explain the decrease in drug acetylation observedin patients with CRF. As shown for other major liver enzymes,the results of this study emphasize that uremic serum containsmediators that downregulate hepatic protein synthesis. PTH seemsto be a major culprit in hepatocellular dysfunction in CRF.
Experimental Model
Male Sprague-Dawley rats (Charles River, Saint-Charles, PQ,Canada) that weighed 200 to 300 g were housed in the ResearchCenter animal care facility and maintained on Harlan Tekladrodent diet (Harlan Teklad Global Diets, Montreal, PQ, Canada)and water ad libitum. All of the experiments were conductedaccording to the Canadian Council on Animal Care guidelinesfor care and use of laboratory animals and under the supervisionof our local animal care committee.
CRF was induced by two-stage five-sixths nephrectomy as previouslyreported.15 Control rats were pair-fed the same quantity ofrat chow that was ingested by the rats with CRF on the previousday. At day 41 after the nephrectomy, the rats were housed inmetabolic cages and urine was collected for 24 h to determinethe clearance of creatinine. Rats were killed by decapitationat 42 d after nephrectomy. Blood and tissues were stored at–80°C up to analysis.
Total PTX was performed, as described previously,17,37 7 d beforethe first nephrectomy. For avoidance of hypocalcemia, rats werethen supplemented in calcium by addition of calcium gluconateto drinking water (control 2.5%; CRF 5%). Control rats receivedsham surgery in the neck region.
Primary Hepatocyte Culture
Hepatocytes were isolated from normal rats according to themodified two-step liver perfusion method of Seglen as previouslypublished.29 Collagenase type 4 (Worthington, Lakewood, NJ)was used. Isolated hepatocytes were plated and cultured in 2ml of William E medium containing 10% of serum from rats withCRF or from control animals. The serum of one rat was used forone experiment. Hepatocytes were incubated during 48 h and werethen harvested by scraping. Samples were stored at –80°Cup to analysis.
To assess whether liver Nat2 could be downregulated by PTH,we measured the ability of rat synthetic 1-34 PTH (Sigma-Aldrich)to depress the Nat of normal hepatocytes. The concentrationof PTH used was 10–9 M. Incubation time was 48 h (mediumwas changed completely after 24 h to maintain PTH concentration).To determine whether NF-B pathway could be involved in the effectof PTH on Nat2, we blocked this pathway using andrographolide(Calbiochem, San Diego, CA), a widely known NF-B inhibitor.42
Liver Cytosol Preparation
Cytosols were prepared according to the method of Schneck etal.43 with slight modifications.
Western Blot Analysis
Nat1 and Nat2 were assessed by Western blotting according toa previously published procedure.44,45 Rat Nat2 was detectedusing a specific polyclonal rabbit antiserum, raised againstthe C-terminal dodecapeptide common to human NAT1 and mouseand rat Nat2 as described previously.46 β-Actin was detectedusing a mouse anti-chicken β-actin (Neo-Markers, Fremont,CA). Immune complexes were revealed by peroxidase-conjugatedAffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch Laboratories,West Grove, PA) and goat anti-mouse IgG coupled to peroxidase(Sigma-Aldrich), respectively.
Rat Nat1 was detected using rabbit anti-rat antibodies recognizingboth Nat1 and 2. For achievement of a specific quantificationof rat Nat1, the Nat2 isoform is immunoprecipitated before SDS-gelelectrophoresis. Cytosols were diluted 1:1 with IP buffer pH7.4 (300 mM NaCl, 20 mM Tris, 2 mM EDTA, 2 mM EGTA [pH 8], and0.1% Tween 20) to which Complete miniprotease inhibitor cocktail(Roche) was added. Diluted cytosols were incubated with thesame amount of prewashed Protein A Sepharose (GE HealthcareBio-Sciences, Baie d'Urfé, PQ, Canada) for 10 min at4°C and then centrifuged for 1 min to pellet the beads.The supernatant was subsequently incubated with anti-Nat2 for2 h at 4°C. Fresh Protein A Sepharose CL-4B was added tothe samples, and incubation was continued overnight at 4°C.Sepharose beads were removed by centrifugation, and supernatantswere frozen up to analysis of rat Nat1 by Western blot.
RNA Isolation and Real-Time Quantitative PCR Analysis
Total RNA were extracted from liver and hepatocytes by usingthe RNeasy Midi and Mini Kit (Qiagen, Mississauga, ON, Canada),respectively. Specific primer and probe sets for rat Nat1 and247 and for glyceraldehydes-3-phosphate dehydrogenase48 wereused for quantitative PCR analysis. Resulting data were processedby the delta Ct method.49
Evaluation of Nat2 Activity
The metabolic activity of rat Nat2 in either treated hepatocytesor cytosolic liver preparation of the different groups of ratswas determined on the basis of the method described by Andreset al.,50 which has been modified by Mattano and Weber.51 Themethod measures the production of N-acetyl-PABA while samplesare incubated with PABA. Controls and samples were run in triplicate.
Evaluation of Rat PTH
PTH was measured by using the Rat intact PTH ELISA Kit (AlpcoDiagnostics, Salem, NH), which measures the intact 1-84 PTH.The lowest detectable level is 1.6 pg/ml.
Statistical Analysis
The results are expressed as means ± SEM. Differencesbetween groups were assessed by using either unpaired t testor an ANOVA test followed by Scheffe post hoc comparison. Thethreshold of significance was P < 0.05.
This work was supported by the Canadian Institute of HealthResearch and Fonds de la Recherche en Santé du Québec.V.P. is a scholar of the Fonds de la Recherche en Santédu Québec. Edith Sim thanks the Wellcome Trust for financialsupport.
Part of this work was presented at the 14th annual meeting ofthe North American International Society for the Study of Xenobiotics;October 22 to 26, 2006; Rio Grande, Puerto Rico.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Introduction
RESULTS
B, as previously shown for CYP450.17 Addition of 30 µM andrographolide to hepatocytes cultured in the presence of 10–9 M PTH (n = 6) prevented the decrease in Nat2 gene expression induced by PTH with a subsequent effect on Nat2 protein level (Figure 7). 
) and CRF (
) rats. Control results were defined as 100%. Data are means ± SEM. Representative blots are shown in insert. *P < 0.001 versus CTL rats.
) in CTL and CRF rat liver with or without PTX. CTL rats were defined as 100%. Data are means ± SEM. Representative blots are shown in insert. 
