The simultaneous inhibition of SLC6A19 and BCRP transporters leads to an increase of indoxyl sulfate (a uremic toxin) in plasma and kidney

Abstract

SLC6A19 inhibitors are being studied as therapeutic agents for Phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7{plus minus}7.7 compared to vehicle 0.3{plus minus}0.1 (P=0.01) as determined by tissue mass spectrometry imaging (tMSI) analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney transporters OAT1, OAT3 and BCRP. RA836 inhibits BCRP with an IC₅₀ of 0.045 µM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using LC-MS. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, while compounds inhibiting both transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma.