Quantification of Urinary Inflammatory Markers

The inflammatory cytokines, chemokines, and neurotrophins for investigation in urine samples were similar to those in our previous study [24 (link),25 (link)]. The targeted analytes in urine were assayed using commercially available microspheres with the Milliplex^® Human cytokine/chemokine magnetic bead-based panel kit (Millipore, Darmstadt, Germany). A total of 11 targeted analytes included Eotaxin, IL-6, IL-8, C-X-C motif chemokine ligand 10 (CXCL10), MCP-1, macrophage inflammatory protein (MIP)-1β, regulated upon activation, normal T-cell expressed and presumably secreted (RANTES), and TNF-α measured with the multiplex kit catalog number HCYTMAG-60K-PX30; NGF was measured with the multiplex kit catalog number HADK2MAG-61K and BDNF was measured with the multiplex kit catalog number HNDG3MAG-36K. The following laboratory procedures for the quantification of these targeted analytes were performed similarly to those in our previous study [30 (link)]. The urinary PGE2 level was measured using a high-sensitivity ELISA kit (Cayman, MI, USA), according to the manufacturer’s instructions. The detailed procedures were similar to those reported in a previous study [44 (link)].