Materials and Methods

Experimental Model.

Male C57BL/6 mice (Charles River Laboratories, Hollister, CA), weighing 20 to 25 g, were housed in the Research Centre animal care facility and maintained on a Teklad diet (Harlan Laboratories Inc., Montreal, QC, Canada) and water ad libitum. The animals were given an acclimatization period of at least 7 days before any experimental work was done. All of the experiments were conducted according to the Canadian Council on Animal Care guidelines for care and use of laboratory animals. Our animal facilities are approved by the Canadian council for animal care. Studies were performed using two groups of mice: CRF (n = 12) and control (n = 12). Chronic renal failure was induced by two-stage 3/4 nephrectomy. To do so, we adapted the 5/6 nephrectomy model (Leblond et al., 2001, 2002) commonly used for the rat with slight modifications. In brief, a dorso-lumbar incision was made and apexes (two quarters) of the left kidney were removed with a scalpel on day 1. A second dorso-lumbar incision was made on the opposite side on day 15, and a right total nephrectomy was performed. After surgery, CRF animals were fed mouse chow and water ad libitum. Mice from the control group underwent two sham laparotomies (day 1 and 15). Body weight was measured every other day for the duration of the study. Control mice were pair-fed the same amount of mouse chow that was ingested by the CRF mice on the previous day. On day 49, 5 weeks after the total nephrectomy, mice were sacrificed by decapitation. Sera were stored at −80°C for the measurement of serum creatinine and urea.

Preparation of Liver Microsomes and Cytosols.

Mouse livers were immediately excised after death, and microsomes were isolated by differential centrifugation (Tindberg et al., 1996) as described previously (Leblond et al., 2001). The pellets containing the microsomes were stored at −80°C in 0.1 M Tris (pH 7.4), 20% glycerol, and 10 mM EDTA until analysis. Microsomal protein content was measured using the Micro BCA Protein Assay, using bovine serum albumin as standard protein (Pierce, Rockford, IL). Cytosolic proteins were dosed using the Bradford method (Coomassie Plus Protein Assay Reagent), with bovine serum albumin as standard (Pierce).

Western Blot Analysis.

Protein expression of the major drug-metabolizing enzymes was analyzed by Western Blot. Mouse CYP1A2, CYP2C29, and CYP2E1 were detected using polyclonal goat anti-rat 1A1, 2C11, and 2E1 (Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan), respectively. CYP2D was detected by using a rabbit anti-human 2D (Oxford Biochemical Research Inc., Oxford, MI), CYP3A11 was detected by using a polyclonal goat anti-mouse 3A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and NAT2 was detected by using specific polyclonal rabbit anti-rat NAT2 as described previously (Stanley et al., 1996; Simard et al., 2008). β-Actin was detected by using a mouse anti-chicken β-actin (NeoMarkers, Fremont, CA). Immune complexes were revealed by corresponding secondary antibodies: swine anti-goat IgG (BioSource International, Camarillo, CA), donkey anti-goat IgG (US Biological, Swampscott, MA), goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), and goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) coupled to peroxidase and the Luminol derivative of Lumi-Light Western blotting substrate (Roche Diagnostics, Laval, QC, Canada).

mRNA Isolation and Real-Time Polymerase Chain Reaction Analysis.

Liver RNA extractions were done by using the RNeasy Mini Kit (QIAGEN, Mississauga, ON, Canada). One microgram of total RNA was used to synthesize cDNA by reverse transcription using Omniscript RT Kit (QIAGEN) and random primers (Invitrogen, Burlington, ON, Canada). Multiplex quantitative polymerase chain reaction (PCR) analysis was performed using an iCycler Real-Time PCR Detection System (Bio-Rad Laboratories, Mississauga, ON, Canada) with Bio-Rad's IQ Multiplex Powermix. Specific primers for Cyp3a11 and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were published previously (Mikula et al., 2005; Richardson and Morgan, 2005). Probe for Cyp3a11 was dye-labeled with FAM, whereas probe for Gapdh was dye-labeled Yakima Yellow. For Cyp1a2, Cyp2c29, Cyp2d22, Cyp2e1, and Nat2, the primer-probe sets used were predesigned TaqMan Gene Expression Assays obtained from Applied Biosystems (Foster City, CA). PCR conditions were optimized to 95°C for 15 s and 60°C for 60 s. The resulting data were processed using the ΔC_t method (Livak and Schmittgen, 2001).

Ex Vivo Metabolism of Erythromycin.

To evaluate the metabolic activity of CYP3A in liver microsomes, erythromycin N-demethylation was determined as described elsewhere (Wang et al., 1997; Guevin et al., 2002; Michaud et al., 2005). In brief, 1 mM erythromycin (Sigma-Aldrich) was incubated with 1.5 mg of mouse liver microsomal proteins (either from control or CRF) at 37°C for 30 min in the presence of an NADPH-generating system consisting of the following: 20 mM glucose 6-phosphate, 2 mM NADP, and 0.14 units/ml glucose-6-phosphate dehydrogenase (Sigma-Aldrich) in a total volume of 0.5 ml. The resulting production of formaldehyde was measured by the Nash (1953) reaction and read on a spectrophotometer at 405 nm.

Blood Chemistries and Statistical Analysis.

Blood chemistries (urea, creatinine) were determined with an Architect C1600 clinical analyzer (Abbott, Saint-Laurent, QC, Canada). The results are expressed as mean ± S.E.M. Differences between groups were assessed by using an unpaired t test. The threshold of significance was p < 0.05.