Transport and metabolism of glutathione conjugates of menadione and ethacrynic acid in confluent monolayers of rat renal proximal tubular cells

Abstract

Confluent monolayers of primary rat renal proximal tubular (RPT) cells were used to compare transepithelial transport and concomitant metabolism of two different glutathione (GSH) S-conjugates. For the GSH-conjugated quinone compound, [ 35S]GSH-conjugated menadione (MGNQ), no specific transepithelial transport was observed. Most likely, [ 35S]MGNQ passed the monolayer via paracellular leakage as the result of a reduction in monolayer integrity due to toxicity via extensive redox cycling of the quinone under the culture conditions. RPT cell monolayers metabolise MGNQ into a cysteinylglycine conjugate, which after intramolecular cyclization yields 2 H-(3-glycinyl)-9-hydroxy-10-methyl-1,4-naphthothiazine. Acivicin, an inhibitor of γ-glutamyltranspeptidase, inhibited the formation of this 1,4-napthothiazine adduct. The second product formed is 1,4-napthothiazine formed by loss of glycine via the action of dipeptidases. Similarly, no basolateral (B) to apical (A) transport of a GSH-conjugated α,β unsaturated ketone, [ 14C]ethacrynic acid (EASG), occurred. However, net transport of [ 14C] radioactivity could be observed from A ⇒ B direction. After 8 h, 23% of total [ 14C] radioactivity was transported from the apical to the basolateral chamber. In both the apical and basolateral chambers, free, unconjugated ethacrynic acid (EA) was observed. γGT-mediated metabolism of EASG to the much more unstable cysteinylglycine conjugate leads to relatively large amounts of free EA. Thus, the GSH conjugate is not transported but rather the cysteine adduct and/or free, unconjugated EA. In agreement with this, acivicin reduced A ⇒ B transport of EASG and inhibited the formation of free EA. In conclusion, the confluent monolayers of RPT cells do not or no longer possess active basolateral transport systems for GSH conjugates. However, they are still quite useful for studying biotransformation reactions of thioether conjugates.