The cyclic nucleotides cAMP and cGMP play key roles in cellular signaling and the extracellular regulation of fluid balance. In the kidney, cAMP is excreted across the apical proximal tubular membrane into urine, where it reduces phosphate reabsorption through a dipyridamole-sensitive mechanism that is not fully understood. It has long been known that this cAMP efflux pathway is dependent on ATP and is inhibited by probenecid. However, its identity and whether cGMP shares the same transporter have not been established. Here the expression, localization, and functional properties of human multidrug resistance protein 4 (MRP4) are reported. MRP4 is localized to the proximal tubule apical membrane of human kidney, and membrane vesicles from Sf9 cells expressing human MRP4 exhibit ATP-dependent transport of [³H]cAMP and [³H]cGMP. Both probenecid and dipyridamole are potent MRP4 inhibitors. ATP-dependent [³H]methotrexate and [³H]estradiol-17β-d-glucuronide transport by MRP4 and interactions with the anionic conjugates S-(2,4-dinitrophenyl)-glutathione, N-acetyl-(2,4-dinitrophenyl)-cysteine, α-naphthyl-β-d-glucuronide, and p-nitrophenyl-β-d-glucuronide are also demonstrated. In kidneys of rats deficient in the apical anionic conjugate efflux pump Mrp2, Mrp4 expression is maintained at the same level. It is concluded that MRP4 is a novel apical organic anion transporter and the putative efflux pump for cAMP and cGMP in human kidney proximal tubules.

Methotrexate (MTX) has been used in combination with nonsteroidal anti-inflammatory drugs (NSAIDs) in the treatment of inflammatory diseases as well as malignancies. Especially at high MTX dosages, severe adverse effects with this combination may occur, usually resulting from an impaired renal elimination. It has been shown that the mechanism of this interaction cannot be fully attributed to inhibition of basolateral MTX uptake in renal proximal tubules. Here, we studied the effect of various NSAIDs on MTX transport in membrane vesicles isolated from cells overexpressing the proximal tubular apical efflux transporters human multidrug resistance protein (MRP) 2/ABCC2 and MRP4/ABCC4. MTX was transported by MRP2 and MRP4 with K_m values of 480 ± 90 and 220 ± 70 μM, respectively. The inhibitory potency of the NSAIDs was generally higher against MRP4- than MRP2-mediated MTX transport, with therapeutically relevant IC₅₀ values, ranging from approximately 2 μM to 1.8 mM. Salicylate, piroxicam, ibuprofen, naproxen, sulindac, tolmetin, and etodolac inhibited MRP2- and MRP4-mediated MTX transport according to a one-site competition model. In some cases, more complex interaction patterns were observed. Inhibition of MRP4 by diclofenac and MRP2 by indomethacin and ketoprofen followed a two-site competition model. Phenylbutazone stimulated MRP2 and celecoxib MRP4 transport at low concentrations and inhibited both transporters at high concentration. Our data suggest that the inhibition by NSAIDs of renal MTX efflux via MRP2 and MRP4 is a potential new site and mechanism contributing to the overall interaction between these drugs.