148. The Transgene Cassette Is Not Fully Responsible for Alterations of Renal Cytochrome P450 Expression after Systemic Administration of Recombinant Adenovirus

Abstract

Gene transfer to the kidney has shown promise in treating severe renal disease and prevention of acute renal graft rejection in several animal models. To date, recombinant adenovirus (Ad) is the most efficient vector for gene delivery to the kidney. Although Ad does not infect the kidney efficiently when administered systemically, transgene products secreted from the liver can reach the kidney at therapeutic levels. We have found that systemic administration of recombinant Ad significantly alters the expression of the cytochrome P450 (CYP) enzymes, largely responsible for the clearance of toxicants and the metabolism of xenobiotics and endogenous compounds, in the kidney. In order to determine if the transgene cassette could be partially responsible for this effect, Sprague-Dawley rats received one of four treatments via a surgically implanted catheter: 0.5 ml of phosphate-buffered saline (control) or 5.7|[times]|1012 viral particles/kg of a first generation Ad expressing either E.coli beta-galactosidase (AdlacZ), murine erythropoietin (Epo) or no transgene (Null). Animals were sacrificed 0.25, 1, 4 and 14 days after injection. Renal CYP protein expression and mRNA levels were analyzed by Western blot and RT-PCR. All treatments significantly induced protein levels of the CYP4A family. Following AdlacZ administration, CYP4A increased by 50%, 32% and 64% above control at 0.25, 1 and 4 days, respectively (p<0.01). The vector expressing Epo raised CYP4A protein levels by 42%, 24% and 64% and the Null vector produced elevations of 74%, 25% and 49% above the control at the same timepoints (p<0.01). CYP4A protein expression recovered by 14 days in each group. RT-PCR revealed that mRNA levels of CYP4A2, the primary CYP4A isoform responsible for renal fatty acid metabolism, increased by 16.8% and 19% above control 24 hours after administration of AdlacZ and Epo, respectively (p<0.01). CYP4F1, an isoform responsible for metabolism of fatty acids, prostaglandins and leukotrienes, was unaffected in all treatment groups. In contrast, renal CYP2E1, which metabolizes small lipophilic compounds and carcinogenic agents, was significantly reduced throughout the study. CYP2E1 mRNA levels in animals treated with AdlacZ were 74%, 78%, 69% and 66% of control at 0.25, 1, 4 and 14 days, respectively (p<0.01). Similar reductions were seen with Epo and Null vectors although levels form animals treated with Epo began to recover by 14 days. Real time quantitative PCR revealed that viral genomes (|[sim]|850/100 ng DNA) were initially present in the kidney and declined throughout the study for all treatments. These results suggest that systemic administration of Ad significantly altered the transcription and translation of several key CYP enzymes in the kidney regardless of the transgene expression cassette. This should be considered for accurate drug monitoring when employing recombinant Ad vectors in the clinic. Additional mechanistic studies assessing the effect of expression of Ad early genes and local and systemic eicosanoid biosynthesis on the expression of renal CYP will allow further explanation of these results.