Active, na-lndependent, organic anion secretion in killifish renal proximal tubules

Previous studies with p-aminohippurate (PAH, Mw 194) and fluorescein (FL, Mw 332), showed that renal secretion of organic anions is driven by indirect coupling to sodium. Here we used killifish proximal tubules and laser scanning confocal microscopy to study the uptake and secretion of a larger organic anion, fluorescein-methotrexate (FL-MTX, Mw 925). When tubules were incubated in medium containing 2 (lM FL-MTX, dye accumulated in both cells and tubular lumen. At steady-state, luminal fluorescence was 4-5 times higher than cellular fluorescence. In contrast to FL, pretreatment of the tubules with 0.1 mM ouabain did not affect cellular or luminal FL-MTX, and replacement of medium sodium by N-methylglucamine had only a small effect; preincubation with glutarate had no effect. Thus, a substantial component of the uptake and secretion of FL-MTX is Na-independent. Transport of FL-MTX was inhibited by micromolar concentrations of other organic anions (MTX, folate, probenecid, bromcresol green, bromphenol red, leukotriene C4), but 1 mM PAH had only a small effect. In contrast, PAH completely blocked FL transport. Surprisingly, FL-MTX secretionwas reduced by cyclosporine A and verapamil, agents that block p-glycoproteinmediated transport, but do not affect FL transport. In conclusion, FL-MTX is secreted by a distinct Na-independent mechanism. Both the basolateral and luminal steps differ from those usually associated with FL and PAH secretion. (Supported by Dutch Kidney Foundation grant C.90.1047).