The effect of chronic kidney disease on CYP2B expression and activity in male Wistar rats

Abstract Chronic kidney disease (CKD) is characterized by progressive reduction in kidney function over time. CKD affects greater than 10% of the population and its incidence is on the rise due to the growing prevalence of its risk factors. Previous studies demonstrated CKD alters nonrenal clearance of drugs in addition to reducing renal clearance. We assessed the function and expression of hepatic CYP2B enzymes using a rat model of CKD. CKD was induced in Wistar rats by supplementing their chow with adenine and confirmed through the detection of elevated uremic toxins in plasma. Liver enzymes AST and ALT were unchanged by the adenine diet. Bupropion was used as a probe substrate for hepatic CYP2B function using rat liver microsomes. The resulting metabolite, hydroxy‐bupropion, and bupropion were quantified by ultra‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry. Level of mRNA and protein were determined by RT‐PCR and Western blot, respectively. The results of our study demonstrate that CYP2B1 is downregulated in a rat model of CKD. CYP2B1 mRNA level was significantly decreased (88%, P < 0.001) in CKD relative to control. Similarly, maximal enzymatic velocity (V max) for CYP2B was decreased by 46% in CKD relative to control (P < 0.0001). Previous studies involving patients with CKD demonstrated altered bupropion pharmacokinetics compared to control. Hence, our results suggest that these alterations may be mediated by attenuated CYP2B hepatic metabolism. This finding may partially explain the alterations in pharmacokinetics and nonrenal drug clearance frequently observed in patients with CKD.


| INTRODUC TI ON
Chronic kidney disease (CKD) affects greater than 10% of the population and its prevalence has been on the rise due to an increased incidence of common risk factors such as diabetes mellitus and hypertension. 1,23,35 CKD is defined as a progressive loss of renal function or significant pathological changes to renal structure which have been present for more than 3 months. 15,26,34 A major clinical feature of this disease is the decline in glomerular filtration rate (GFR) due to kidney damage. Since the kidneys are a major site for xenobiotic processing and elimination, the impact of CKD extends beyond the primary function of the kidneys by dramatically altering drug clearance. 39 Chronic kidney disease patients are prescribed a mean of 14.2 different drugs concurrently to manage their underlying condition as well as associated comorbidities such as diabetes, hypertension, and cardiovascular disease. 40 This combination leads to an increased incidence of adverse drug events due to drug interactions and altered drug pharmacokinetics. 20 Dosing alterations for some renally cleared drugs in CKD patients are fairly well characterized; however, nonrenal metabolism and transport have also been shown to be altered in CKD. 8,24,[27][28][29]37,38,45 Explanations for this focus on alterations in transcriptional, translational, and nuclear regulation of hepatic or enteric drug metabolizing enzymes and transporters, which may be caused by increased levels of inflammatory cytokines and uremic toxins. 5,17,30,44,45 The liver is the primary site for drug metabolism due mostly in part to the cytochrome P450 (CYP) superfamily of enzymes.
Previous studies investigating changes of nonrenal clearance in CKD used animal models to demonstrate differences in function or expression of rat CYP1A2, CYP2C6, CYP2C11, CYP2D1/CYP2D2, CYP3A1, CYP3A2, CYP4A1/CYP4A3, as well as changes in the epigenetic regulation of CYP2C11 and CYP3A2. 9,16,18,24,32,[43][44][45] This study focused on CYP2B isoenzymes since they have been minimally studied in the context of CKD despite having a clinically relevant role in the metabolism of many drugs, nutraceuticals, and herbal medicines. Specifically, the CYP2B6 enzyme alone is responsible for metabolism of 7.2% of prescribed medications (W. 33,51 Human CYP2B6 accounts for 6% of the total hepatic P450 content but has high interindividual and ethnic variation in expression. For example, 85% of Caucasians have detectable CYP2B expression in contrast to only 30% in a study from a Japanese population. 2,21,36 CYP2B6 shares approximately 75% sequence homology with rat CYP2B1 and CYP2B2. 6 Rat CYP2B1 and CYP2B2 are isoenzymes with 97% primary structural identity overlap and have similar substrate specificities. 48 Catalytic activity is the primary differentiator between the two enzymes, with CYP2B1 being more active. 31,48 This is highlighted in a study that observed the metabolism of a CYP2B selective marker, bupropion (BUP), by rat liver microsomes. The paper reported that 75% of the BUP conversion to its metabolite OH-bupropion (OH-BUP) was attributable to CYP2B1, while CYP2E1 and CYP2C11 accounted for 10.9% and 8.7%, respectively. 31 In humans 71% of the OH-BUP formation is due to CYP2B6 and multiple studies have reported reductions in conversion of BUP to OH-BUP in CKD patients. 4,7,12,14,42 To date, no study has assessed the expression of CYP2B6 or the expression and function of rat CYP2B1 and CYP2B2 in CKD. Using a model of CKD induced by an adenine diet in male Wistar rats, we investigated the expression and function of CYP2B1 and CYP2B2.

| Rat model of CKD
The adenine model of CKD used was developed by adhering to a previously described protocol. 8,46 Male Wistar rats (150 g) were acquired from Charles River Laboratories and allowed a 5-day acclimation period before commencement of the study diet. Animals were randomly divided into control and CKD groups (n = 9 and n = 12, respectively). Animals allocated to the CKD group were given a diet supplemented with 0.7% adenine for 5 weeks, followed by 3 weeks of control diet. Control animals were pair-fed to the CKD group using standard rat chow for the entire study. Animals were euthanized using isoflurane as a general anesthetic, followed by subsequent decapitation at the conclusion of the study. Plasma and liver tissue were harvested from the animals. Samples were kept frozen at −80°C and −20°C for liver and plasma, respectively, until analysis. Animal protocols were approved by the University of Western Ontario Animal Care Committee.

| qPCR
Total RNA extraction was completed by using TRIzol reagents following manufacturers instructions (Life Technologies, Waltham, MA, USA). Spectrophotometry was used to assess quality and quantity of RNA. Synthesis of cDNA was performed from total RNA using qScript cDNA supermix, which contained reverse transcriptase. The qPCR reaction mix was prepared in ac-

| Western blot
To assess expression of CYP2B1 and CYP2B2, hepatic microsomes were isolated as previously described. 45 Twenty micrograms of protein per sample was electrophoresed on a 10% polyacrylamide gel containing 0.1% SDS. The microsomal proteins were subsequently transferred to nitrocellulose in preparation for immunoblotting. Bands at 55 and 56 kDa represented CYP2B1 and CYP2B2. Due to their close proximity and difficulty with satisfactory separation, both the 55 and 56 kDa bands were combined to produce the results on CYP2B expression. A common band was found between 60 and 62 kDa but was not included in densitometry analysis.

| Microsomal metabolism
Microsomes were aliquoted in duplicate at a concentration of 0.5 mg/mL into 96-well plates along with microsomal incubation buffer (50 mmol/L KH2PO4 and 5 mmol/L MgCl2 at pH = 7.4).
Bupropion was also added to the reaction at concentrations ranging from 10 to 500 μmol/L. NADPH at a final concentration of 1 mmol/L was added to commence the reaction, which proceeded with linear kinetics for 30 minutes. The reaction was stopped using ice-cold acetonitrile (ACN), at a 3:1 ratio of ACN to sample. The solution contained 40 ng/mL of flurazepam to serve as an internal standard.
Subsequently the samples were centrifuged at 3000 rpm for 5 minutes before being diluted 1:5 in water and subjected to mass spectrometry for quantification.

| Hydroxy-bupropion UPLC-MS quantification
A standard curve was generated by serially diluting bupropion and hydroxy-bupropion from 50 μmol/L to 50 nmol/L. Five microliters of sample was injected onto an Acquity BEH C18 column

| Data and statistical analysis
Statistical analysis was completed by unpaired, two-tailed t tests to compare control and CKD groups. Data are displayed as mean ± standard error of the mean. The threshold for significance was P < 0.05 except where several metabolites were tested simultaneously in which case Bonferroni correction was applied. Statistics and graphs were generated using GraphPad Prism version 6.0 software. 25 In regard to the microsomal metabolism assays, results were plotted using Michaelis-Menten enzyme kinetics to calculate the metabolic parameters K m (the Michaelis constant) and V max (maximum velocity).

| RE SULTS
Kidney function was assessed at the end of the study by quantification of plasma urea and creatinine ( Figure 1). Both plasma urea and creatinine were significantly elevated in the CKD group compared to control (P < 0.0001), evident by an 8.7 and 9.1-fold (57.7 ± 7.4 and 6.6 ± 0.5 mmol/L; 208.2 ± 20.4and 22.8 ± 0.6 μmol/L) increase in concentration, respectively.
To ensure that the adenine diet did not produce overt liver toxicity, liver function was assessed by determination of plasma levels of the hepatic enzymes AST and ALT (Figure 2). No significant difference was seen between control and CKD groups for either AST or ALT (P = 0.354 and P = 0.308, respectively). CYP2B1 and CYP2B2 mRNA levels from CKD and control groups were quantified using qPCR (Figure 4). Levels of CYP2B1 mRNA were markedly lower in the CKD group compared to control, accounting for an 87% decline in expression (P < 0.001). No statistically significant differences in expression were seen for CYP2B2 between groups (P = 0.608).
Protein expression of CYP2B showed no significant differences between control and CKD groups ( Figure 5, P = 0.847).
The rate of metabolism of bupropion by rat liver microsomes is shown in Figure 6. Rat liver microsomes from the CKD group showed significantly decreased metabolism of bupropion compared to control (P < 0.05). Michaelis-Menten analysis showed dramatically higher K m (5.2 fold) as well as significantly lower V max (46% decline) and intrinsic clearance (88% decline) in the CKD group relative to control (P < 0.0001) ( Table 1).

| D ISCUSS I ON
To our knowledge, the expression and function of CYP2B1 and CYP2B2 has not been previously evaluated in the setting of CKD.
Although CYP2B1 and CYP2B2 are rat-specific enzymes, there is a high sequence homology with human CYP2B6. 22 The importance of CYP2B6 in human drug metabolism is emphasized in the literature F I G U R E 1 Plasma urea (A) and creatinine (B) levels in control and CKD Wistar rats, measured in mmol/L and μmol/L, respectively. Rats were fed standard chow (control) or standard chow with 0.7% adenine (CKD) for 42 days. Results are presented as mean ± SEM, n = 10-12 per group and the threshold for significance after Bonferroni correction was 0.025, ****P < 0.0001 compared to control F I G U R E 2 Plasma AST (A) and ALT (B) levels in control and CKD Wistar rats, measured in U/L. Rats were fed standard chow (control) or standard chow with 0.7% adenine (CKD) for 42 days. Results are presented as mean ± SEM, n = 10-12 per group and the threshold for significance after Bonferroni correction was 0.025 (P = 0.354 and P = 0.308, respectively) due to its contribution to the biotransformation of drugs such as cyclophosphamide, bupropion, clopidogrel, and efavirenz 2,13,36 ; H-J. 3,10,22,50 The in vivo model of CKD was developed using a 0.7% adenine diet in male Wistar rats. Measurement of plasma urea and creatinine were 8.7 and 9.1-fold higher in the CKD group, respectively, which validated the presence of renal damage in the model. In addition, plasma concentration of the uremic toxins indoxyl sulfate, p-cresyl sulfate, hippuric acid, and phenyl sulfate were also significantly elevated in the CKD group. This difference in plasma uremic toxins supports that the 0.7% adenine supplemented rats had decreased renal function and that their plasma resembled the uremic milieu seen in CKD patients. Previous model validation and results from the current study reported no increase in liver function enzymes AST or ALT in CKD animals compared to control. 8,46 Accordingly, the observed changes in liver CYP2B1 expression and function are not attributed to direct hepatic damage. A significant change in mRNA level of CYP2B1 was observed as mRNA levels in CKD animals were decreased by 87.3% relative to control. The mRNA level of CYP2B2 was unchanged between CKD and control. Follow-up experiments to assess protein expression of CYP2B showed no difference between disease and control. This result may be due to nonspecific antibody binding, since CYP2B1 and CYP2B2 share 97% sequence homology.
Based on this, the unchanged CYP2B protein level between control and CKD may not accurately depict CYP2B1 expression, which was unable to be determined by Western blot. To determine the effect of CKD on CYP2B1 protein level, future studies using mass spectrometry to specifically quantify CYP2B1 concentration are required.
Although overall CYP2B protein expression appeared unchanged, the pronounced decrease in mRNA expression spurred further exploration into the metabolic activity of CYP2B1 as it is noted to be more catalytically active than CYP2B2. 31 BUP was selected as the probe substrate for CYP2B1 due to previous work validating its high metabolism by this enzyme. 31 A significant decrease was observed in the function of CYP2B1 in CKD compared to control, which was exemplified by reductions in V max and intrinsic clearance by 46.2% and 88.4%, respectively.
The decrease of BUP conversion to OH-BUP in CKD rats supports the mRNA data since CYP2B1 is the primary metabolic enzyme for this compound. 31 The significant decreases in V max and intrinsic clearance can be explained by the loss of CYP2B1 expression. The marked increase in K m is likely reflective of the activity F I G U R E 3 Concentrations of plasma indoxyl sulfate (A), P-cresyl sulfate (B), hippuric acid (C), and phenyl sulfate (D) in μmol/L. Rats were fed standard chow (control) or standard chow with 0.7% adenine (CKD) for 42 days. Results are presented as mean ± SEM, n = 10-12 per group and the threshold for significance after Bonferroni correction was 0.0125 **P = 0.0045, ***P = 0.0005, † † † P = 0.0003, and ****P < 0.0001 compared to control F I G U R E 4 Relative hepatic mRNA expression of CYP2B1 (A) and CYP2B2 (B) in an adenine diet CKD model. The sample mRNA was extracted from total liver homogenate. Results are presented as mean ± SEM, n = 9-12 per group, and the threshold for significance after Bonferroni correction was 0.025 ***P < 0.001 compared to control. β-actin was used as the housekeeping gene for CYP2B1 and CYP2B2 of other CYP isoenzymes that have lower affinity for bupropion (eg, CYP2C11 or CYP2E1). CYP2B1 normally accounts for 75% of OH-BUP production while CYP2C11 will metabolize approximately 11% of BUP. 31 In our model, CYP2B1 expression is decreased and the activity of CYP2C11 has been shown to decrease significantly in CKD as well. 19,45 A study performed on rat primary hepatocytes which were incubated with serum from CKD patients showed no difference in CYP2E1 expression, which supports this theory. 24 Therefore, we suggest that the production of OH-BUP in microsomes from rats with CKD may be mediated by CYP isoforms, such as CYP2E1 that are unaffected by CKD and have a low affinity for BUP.  14 Furthermore, the pharmacokinetics of BUP were similar in a study that compared BUP metabolism in hemodialysis patients who smoked to a control population with normal renal function. 49 However, there was a much larger AUC for OH-BUP in the endstage renal disease (ESRD) group compared to control, indicating there was accumulation of the drug in these patients. 49 An in vitro study in 2014 showed no change in human microsomal CYP2B6 function when incubated with uremic serum from human ESRD patients pre-and postdialysis. 47 The authors did note some limitations of the study which included a small sample size (n = 10) and an entirely African-American subject base despite no case-control matching incorporated into the study design. Patients from that study also received dialysis three times per week, which may have nullified an inhibitory effect of uremic serum on metabolism due to insufficient time allowed for toxin accumulation. The authors also suggested that their use of ultrafiltered serum may have removed some of the more strongly protein-bound uremic toxins which have been shown to have interactions with CYP enzymes in other studies. 11,41,47 The study by Volpe et al from 2014 also supports the notion that altered CYP2B function is unlikely to be due to direct inhibition from uremic toxins.
F I G U R E 5 (A) Relative protein expression of CYP2B in hepatic microsomes from an adenine model of CKD in Wistar rats. Control and CKD groups are presented with mean ± SEM, n = 9-12 per group. β-actin was used as the housekeeping protein. (B) Representative blots with C = Control, CKD = CKD F I G U R E 6 Bupropion metabolism to OH-bupropion as a measure of CYP2B activity in rat liver microsomes from control and CKD animals (pmol/min/mg protein). V max and K m were determined using the Michaelis-Menten model, n = 9-12 per group Alternatively, studies directed toward altered drug dosing for substrates of CYP2B6 can help to lower the occurrence of subtherapeutic or toxic dosing events in patients with CKD.

ACK N OWLED G EM ENT
This work was supported by grants from the Natural Sciences

and Engineering Research Council and Canadian Foundation for
Innovation.

D I SCLOS U R E S
None declared.