Mlcn20

Urinary creatinine was determined by using a QuantiChrom creatinine assay kit according to the protocol (DICT-500; BioAssay Systems, Hayward, CA, USA). Quantikine Elisa kits were used for measurement of urinary albumin (E90-1134, Bethyl, Montgomery, TX, USA) and urinary Neutrophil gelatinase-associated lipocalin (NGAL) (MLCN20, R&D Systems) according to the manufacturer’s instructions.

Spleen was homogenized in homogenizing buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Sodium deoxycholate), the homogenate was centrifuged and supernatants were used for cytokine measurements using mouse IL-1β ELISA kit (eBioscience, 88-7013-22) according to the manufacturer’s protocols, and read on a Luminex machine. In order to assess intestinal inflammation, fecal samples from day 1, 4, and 7 post-DSS treatment were homogenized in homogenizing buffer, spun down and supernatants were collected and assayed for Lipocalin-2 by ELISA according to manufacturer’s instructions (R&D, MLCN20). Serum endotoxin level was measured using ToxinSensor Chromogenic Endotoxin Assay Kit (GenScript, L00350). Briefly, diluted plasma was incubated with Limulus amoebocyte lysate (LAL) and after performing several reactions under endotoxin free condition; samples were read spectrophotometrically at 545 nm. The plasma endotoxin levels were calculated against a standard curve of endotoxin (E. coli 0113:H10) concentrations of 0.1, 0.05, 0.025, 0.0125 and 0 EU/ml.

Blood was collected in Microtainer tubes (BD) from cardiac puncture of mice under isoflurane, and serum was obtained after centrifugation at 13,000 rpm for 2 min at room temperature. Serum parameters were performed biochemically following the manufacturer’s instructions [blood urea nitrogen (BUN): DIUR-100, Bioassay Systems, Hayward, CA, USA; uric acid: DIUA-250, Bioassay Systems; Creatinine: C753291, Pointe Scientific; fructose: EFRU-100, Bioassay Systems]. Urinary neutrophil gelatinase-associated lipoprotein (NGAL; MLCN20, R&D Systems, Minneapolis, MN, USA), and albumin (Albuwell M; Ethos Biosciences) levels were normalized to units of creatinine. Determination of parameters in tissue was performed in freeze-clamped tissues and measured biochemically following the manufacturer’s protocol [uric acid: DIUA-250, Bioassay Systems; Hydroxyproline: DHYP-100, Bioassay Systems; thiobarbituric acid-reactive substances (TBARS): DTBA-100, Bioassay Systems; and uric acid: DIUA-250, Bioassay Systems].

All mice were anesthetized with Zoletil (5 mg/kg, Virbac Laboratories, Carros, France). Blood samples (n = 8 per group) were extracted transcardially through the apex of the left ventricle with a 1-mL syringe, and the blood was allowed to clot for 2 h at room temperature. After centrifugation, the serum was removed and stored at -80°C until analysis. Serum glucose, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were determined using enzymatic colorimetric assays from Green Cross Reference Laboratory (Yongin-si, South Korea). Serum insulin, leptin, and lipocalin-2 concentrations were measured using insulin (AKRIN-011T, Shibayagi, Gunma, Japan), leptin (AKRLP-011, Shibayagi), and lipocalin-2 (MLCN20, R&D systems, Minneapolis, MN, USA) mouse enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturers’ protocols. The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated by multiplying fasting serum glucose (mg/dL) by fasting serum insulin (mU/ml) and dividing the total by 405.

The abundance of lipocalin-2 in feces was determined by ELISA using mouse lipocalin-2/NGAL detection kit (MLCN20; R&D) according to the manufacturer’s instruction.