Evaluation of P-glycoprotein-Mediated Renal Drug
Study Objective: To evaluate P-glycoprotein (P-gp)-mediated renal drug interactions in an in vitro model of tubular secretion.
Design: In vitro experiment.
Setting: University-affiliated pharmacokinetics laboratory.
Cell Lines: Madin-Darby canine kidney (MDCK), multidrug-resistant-1 (MDR1)-MDCK, and human colon carcinoma (Caco-2) cells.
Intervention: Transepithelial transport (basolateral-to-apical and apical-to-basolateral) of cimetidine was assessed in the absence and presence of various concentrations of the P-gp inhibitors itraconazole and PSC-833 in a renal P-gp cell culture model (MDR1-MDCK).
Measurements and Main Results: Apparent permeability of cimetidine was characterized, and level of P-gp expression was determined by Western blot analysis, in MDCK (wild type), MDR1-MDCK, and Caco-2 cells (for relative comparison). In the presence of PSC-833, cimetidine's apparent permeability value for basolateral-to-apical transport decreased from 2.96 to 1.15 x 10 cm/second, coupled with a decrease in efflux ratio from 2.36 to 1.80. The effect of itraconazole was concentration dependent, with cimetidine's apparent permeability value for basolateral-to-apical transport decreasing from 3.96 to 1.92 x 10 cm/second (p<0.05), resulting in a 50% decrease in efflux ratio. Expression of P-gp was negligible in MDCK (wild-type) cells, but high-level expression was confirmed in both MDR1-MDCK and Caco-2 cells.
Conclusion: P-glycoprotein plays a significant role in the renal tubular secretion of organic cations such as cimetidine, and the high level of P-gp expression in MDR1-MDCK cells makes this a well-suited model for evaluating mechanisms of renal drug interactions.

Evaluation of drug transport mechanisms is becoming increasingly important in drug-delivery and pharmacokinetics research. P-glycoprotein, a plasma membrane-bound efflux protein, initially was identified in cancer cells resistant to a wide variety of chemotherapeutic agents, including anthracyclines, anthracenediones, and vinca alkaloids. Overexpression of P-gp has been associated with drug resistance in various cancers, including solid tumors and hematologic malignancies. During the past decade, P-gp-modulating agents such as PSC-833, verapamil, and cyclosporine have been evaluated clinically as adjunct therapy for multidrug-resistant cancers, with little success. More recently, P-gp has been identified in healthy human tissue such as intestine, liver, brain, and kidney, suggesting that P-gp function may contribute to drug absorption, distribution, and elimination.

The human kidney expresses several basolateral and apical drug transport proteins, including the organic anionic transporter-1, organic cationic transporter (OCT)-1, multidrug resistance-associated protein-2, and P-gp. Renal P-gp is located primarily in glomerular mesangium and the apical membrane of proximal tubule epithelia, where it is likely involved in the elimination of endogenous and exogenous compounds from the systemic circulation. P-glycoprotein-mediated tubular secretion involves transport of substrates across the apical membrane into the tubular lumen. However, the extent of P-gp involvement in the renal elimination of drugs and the effects of drugs and disease states on P-gp function are not clearly understood.

Cimetidine is a histamine₂-receptor antagonist that is widely used to treat gastrointestinal ulcers. It is predominantly excreted unchanged by the kidney, with renal clearance values approximately 4-fold greater than creatinine clearance, indicating extensive tubular secretion. Classified as an organic cation, cimetidine also has been identified as a P-gp substrate based on drug transport studies conducted in multidrug-resistant-1 (MDR1)-Madin-Darby canine kidney (MDCK) cell lines and P-gp-enriched renal brush border membrane vesicles. In an isolated perfused rat kidney model, pretreatment with cimetidine reduced the clearance of rhodamine 123, suggesting that competitive inhibition among P-gp substrates for renal tubular secretion may occur.

Recent studies have demonstrated that P-gp function is inhibited by itraconazole and PSC-833 in vitro. Itraconazole was shown to reverse drug resistance to the P-gp substrates daunorubicin, Adriamycin, and etoposide in P388/ADR cells at concentrations of 0.1-2.8 µg/ml. PSC-833, a nonimmunosuppressive analog of cyclosporine, is one of the most potent P-gp inhibitors in development as an adjunct to cancer chemotherapy. It was shown to inhibit tubular excretion of ivermectin in isolated proximal tubules and significantly reduced the renal clearance of vincristine in a rat model. Taken together, these in vitro and in vivo data suggest that itraconazole and PSC-833 may inhibit renal P-gp at concentrations achieved clinically, resulting in decreased renal tubular secretion. We sought to establish an in vitro model for screening P-gp-mediated renal drug interactions and to evaluate the effects of itraconazole and PSC-833 on the transepithelial transport of cimetidine in MDR1-MDCK cells.