A B S T R A C T Serum and urine from chronically uremic patients and normal individuals were subjected to gel filtration on Sephadex-G10. The effects of the eluted fractions on the uptake of urate and para-aminohippurate by isolated cortical tubules of rabbit kidney were investigated. According to the origin of the samples, one to three major groups of fractions inhibiting both urate and para-aminohippurate transport were disclosed. The first eluted group occurred for all the samples under study. The second one was demonstrated in both sera and urines from uremic patients but only in urines from normal individuals. The third one was exclusively detected in uremic sera and urines. Among all the compounds identified, only hippuric acid, eluted in the fractions of the second group, was capable of inhibiting the uptake of urate and para-aminohippurate in vitro. The concentration for which this inhibitory effect of hippuric acid occurred was in the range of that existing in uremic sera. Indoxyl sulfate, which accumulates to very high concentrations in uremic serum, could not be disclosed in the above-mentioned fractions. This is explained by the strong adsorption of this indole derivative to Sephadex gel. Potassium indoxyl sulfate, when tested in vitro at the concentration existing in uremic serum, substantially inhibited the uptake of both urate and para-aminohippurate. In normal subjects, ingestion of hippuric acid or potassium indoxyl sulfate significantly increased fractional urinary excretion of uric acid. On the basis of these results, it is suggested that progressive retention of hippuric acid, indoxyl sulfate, and other yet unidentified inhibitors may explain the gradual increase in urinary fractional excretion of urate This work was presented in part at a symposium on uremia sponsored by the International

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Characteristics of para-aminohippurate (PAH) transport in the absence of intracellular metabolism were studied with Na+, K+-depleted and ouabain-poisoned rabbit kidney cortex slices. The imposition of a NaCl gradient (out to in) resulted in specific stimulation of PAH uptake. PAH accumulated against its concentration gradient when cell [Na+] was less than medium [Na+]. Conversely, renal cells extruded PAH when cell [Na+] exceeded medium [Na+]. Membrane potential measured with triphenylmethylphosphonium revealed that conditions which created an interior-positive membrane potential inhibited the Na+-dependent transport of PAH but caused stimulation of the Na+-independent component. Characteristics of the Na+-dependent PAH transport system in ouabain-poisoned slices were similar to those previously described in metabolically active tissues.

The effects of K+, Na+, and ouabain on para-aminohippurate (PAH) accumulation by rabbit kidney slices have been further examined. The present studies show that for maximum uptake of PAH to occur in Na+,K+-depleted slices extra-cellular Na+ and K+ ions are required together. Kinetic studies revealed that increasing the external K+ from 0 to 5 mM caused a decrease in the apparent Km of transport. The Vmax values were not changed significantly. In the presence of 5 mM K+, increasing Na+ concentration in the bathing medium from 0 to 145 mM produced an increase in the V max while the apparent Km remained constant. In the presence of 5 mM external K+, ouabain inhibition of PAH uptake was progressively and entirely antagonized by decreasing external Na+. Studies on kidney slices treated with ouabain in a Na+,K+-free medium in order to inhibit active electrolyte transport showed that PAH uptake under anaerobic conditions was specifically enhanced by Na+. This stimulation occurred when cell [Na+] was less than medium [Na+]. This Na+-dependent and energy-independent accumulation of PAH was inhibited by hippuric acid but not by N-methylnicotinamide.