Urinary and Blood-Based Biomarker Analysis

At each visit, 2.5 mL of blood and 50 mL of urine were collected. After collection, the blood was stored in PAXgene Blood RNA tubes, and total RNA was extracted using a PAXgene Blood RNA Kit (PreAnalytiX; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The urine was centrifuged at 2000× g for 20 min, the pellet was transferred and stored at −70 °C until use. Total RNA was extracted from the urinary pellets using a PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Urinary exosomal RNA was isolated from 1 mL of urinary supernatant using spin column-based exoRNeasy serum/plasma midi kits (QIAGEN GmbH, Hilden, Germany). Then, 20 μg of urinary exosomal proteins was separated by SDS-PAGE, transferred to a nitrocellulose membrane, probed with the appropriate primary antibodies, and incubated with horseradish peroxidase-linked secondary antibodies. Blots were visualized with enhanced chemiluminescence detection reagents and quantified using ECL hyperfilm. Band volumes were measured by densitometry in at least three different experiments. Primary antibodies against the following proteins were used: tetraspanin-1 (H00010103, 1:500; Abnova, Taipei, Taiwan) and hemopexin (ab124935, 1:500; Abcam, Cambridge, UK). The details are shown in our previous studies [7 (link),10 (link),11 (link),12 (link),13 (link)].