Indoxyl sulphate

Acetonitrile, creatinine, VMA, HVA, Trp, 5-HIAA and indoxyl sulphate (IS) were purchased from Sigma–Aldrich (St. Louis, MO, USA), DHICA from Toronto Research Chemicals Inc. (Toronto, ON, Canada), formic acid from Riedel-de Haёn (Seelze, Germany). Ultrapure water was prepared by ultrafiltration of distilled water using the Simplicity system (Millipore, Molsheim, France). Mobile phase of 15% Acetonitrile (ACN:H₂O; 15:85) was prepared in deionised water with an addition of 0.05% formic acid. Stock solutions of creatinine, DHICA, VMA, HVA, Trp, 5-HIAA and IS were prepared by diluting given compounds to a concentration of 1 mg/ml in mobile phase. All reagents were of analytical grade.

To mimic uraemic conditions in vitro, EC were treated for 7 days in 75 cm² flasks (NUNC) with standard culture medium supplemented with 20 mmol/L urea (Merck) which roughly corresponds to concentrations seen during chronic renal failure¹⁸ and 375 µmol/L (50 µg/mL) indoxyl sulphate (IS; Sigma‐Aldrich) which corresponds to mean plasma concentrations in uraemic patients.¹⁹ The medium was replaced every 2nd day. Untreated cells served as control.