Phenylmethylsulfonyl fluoride protease inhibitor

Fresh urine was collected up to 2 days before or after the renal biopsy and centrifuged to remove cells and debris. Aliquots were stored at −70°C after the addition of 1 N NaOH (to reach a pH between 7.0 and 8.0) and 10 μl of 0.1 M phenylmethylsulfonyl fluoride protease inhibitor (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). uMCP-1 levels were determined with the Quantikine Human MCP-1/CCL2 Immunoassay kit (R&D Systems, Minneapolis, MN, USA). The uMCP-1 values obtained were normalized to the amount of creatinine in the urine sample and reported as the ratio of uMCP-1 (pg/ml)/urinary creatinine (mg/ml). Urine samples from 10 healthy individuals were used as controls. The urinary biomarker measurements in the controls were performed in the same period used for the patients in this study.

The isolated colons were opened longitudinally and washed with cold PBS, and homogenized in PhosphoSafe Extrction Reagent (Novagen Merck, Darmstadt, Germany) with phenylmethylsulfonyl fluoride protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). The levels of TNF-α and LTB₄ were quantified in colonic tissues using an ELISA performed according to the protocol of the manufacturer. The lower limits of detection for the cytokines were as follows: TNF-α (>5.1 pg/mL; R&D Systems, Minneapolis, MN, USA), and LTB₄ (>13 pg/mL, Cayman Chemical Company, Ann Arbor, MI, USA).