Tures and glue. We immediately resuscitated mice with 1-mL warmed subcutaneous

Tures and glue. We immediately resuscitated mice with 1-mL warmed subcutaneous saline and administered subcutaneous buprenorphine (0.05 lg/g body weight) for pain. Select mice were pretreated with saline, 150 lg NAH, or 5 units heparin (administered via 200 lL subcutaneous injection) 2 h prior to CLP. All assessments of AKI and/or the systemic inflammatory response were performed order Dalfopristin beginning 4 h after CLP/sham surgery.Hemodynamic assessmentWe measured left ventricular pressures and heart rate in open-chest mice. After completion of the experimental protocol, anesthesia was achieved via intraperitoneal ketamine (100 lg/g body weight) and xylazine (15 lg/g body weight). We ventilated mice (MiniVent, Harvard Apparatus, Holliston MA) with 10 lL/g body weight tidal volumes at 120 breaths/min via tracheostomy. We performed a sternotomy and measured left ventricular pressure and heart rate by direct ventricular puncture with a Millar catheter (ADInstruments, Colorado Springs, CO).MethodsMaterials/animalsWe purchased N-desulfated re-N-acetylated heparin (NAH) from Iduron (Manchester, UK) and heparin from Moore Medical (Farmington, CT). We purchased Evans blue dye (EBD), bovine serum albumin (BSA), fluorescein isothiocyanate (FITC)-inulin (Cat # F3272-1G), heparinase-III (H8891), and HS (H7640) from Sigma (St. Louis, MO). We purchased Intramedic PE-10 tubing from Beckton-Dickinson (Franklin Lakes, NJ). We purchased male C57BL/6 mice (8?0 weeks old) from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in standard housing at the University of Colorado Anschutz Medical Center with ad lib access to food and water. Animals of identical age and housing (i.e., littermates) were randomly assigned to experimental or control groups. The Institutional Animal Care and Use Committee of the University of Colorado approved all mouse protocols. The care and handling of animals were in accord with National Institutes of Health guidelines for ethical animal treatment.Serum assaysAfter completion of the experimental protocol, we collected mouse serum via direct cardiac puncture during organ harvest. Blood urea nitrogen (BUN) and serum creatinine were measured by quantitative colorimetric assays (DIUR-500 and DICT-500; BioAssay Systems, Hayword, CA). Serum IL-6, KC, TNF-a, IL-10, and IL-1b were measured via a multiplex immunoassay (K15012C-2; Meso Scale Discovery, Rockville, MD). We measured serum angiotensin II by enzyme-linked immunosorbent j.jebo.2013.04.005 assay (ELISA) after phenyl extraction (400048, Cayman Chemical, Ann Arbor, MI), as per the manufacturer’s instructions (589301, Cayman).Urine assaysAfter completion of the experimental protocol, urine was collected beginning 2 h prior to organ harvest. jmir.6472 Urine pro-2013 | Vol. 1 | Iss. 6 | e00153 Page?2013 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.M. I. Lygizos et al.Heparanase Mediates Early Septic Renal Dysfunctiontein (#500; Bio-Rad, Hercules, CA) and creatinine (BioAssay Systems, as above) were measured via colorimetric assays. We measured urine HS Nutlin (3a) msds degradation activity (GWBF49F83; GenWay Biotech, San Diego, CA) as previously described (Schmidt et al. 2012). Urinary IL-6 was measured by ELISA (M6000B; R D Systems, Minneapolis, MN). We measured urinary nitric oxide species (NOx) using a colorimetric assay of urine nitrate (NO3? and nitrite (NO2?, as per the manufacturer’s instructions (780001; Cayman). We m.Tures and glue. We immediately resuscitated mice with 1-mL warmed subcutaneous saline and administered subcutaneous buprenorphine (0.05 lg/g body weight) for pain. Select mice were pretreated with saline, 150 lg NAH, or 5 units heparin (administered via 200 lL subcutaneous injection) 2 h prior to CLP. All assessments of AKI and/or the systemic inflammatory response were performed beginning 4 h after CLP/sham surgery.Hemodynamic assessmentWe measured left ventricular pressures and heart rate in open-chest mice. After completion of the experimental protocol, anesthesia was achieved via intraperitoneal ketamine (100 lg/g body weight) and xylazine (15 lg/g body weight). We ventilated mice (MiniVent, Harvard Apparatus, Holliston MA) with 10 lL/g body weight tidal volumes at 120 breaths/min via tracheostomy. We performed a sternotomy and measured left ventricular pressure and heart rate by direct ventricular puncture with a Millar catheter (ADInstruments, Colorado Springs, CO).MethodsMaterials/animalsWe purchased N-desulfated re-N-acetylated heparin (NAH) from Iduron (Manchester, UK) and heparin from Moore Medical (Farmington, CT). We purchased Evans blue dye (EBD), bovine serum albumin (BSA), fluorescein isothiocyanate (FITC)-inulin (Cat # F3272-1G), heparinase-III (H8891), and HS (H7640) from Sigma (St. Louis, MO). We purchased Intramedic PE-10 tubing from Beckton-Dickinson (Franklin Lakes, NJ). We purchased male C57BL/6 mice (8?0 weeks old) from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in standard housing at the University of Colorado Anschutz Medical Center with ad lib access to food and water. Animals of identical age and housing (i.e., littermates) were randomly assigned to experimental or control groups. The Institutional Animal Care and Use Committee of the University of Colorado approved all mouse protocols. The care and handling of animals were in accord with National Institutes of Health guidelines for ethical animal treatment.Serum assaysAfter completion of the experimental protocol, we collected mouse serum via direct cardiac puncture during organ harvest. Blood urea nitrogen (BUN) and serum creatinine were measured by quantitative colorimetric assays (DIUR-500 and DICT-500; BioAssay Systems, Hayword, CA). Serum IL-6, KC, TNF-a, IL-10, and IL-1b were measured via a multiplex immunoassay (K15012C-2; Meso Scale Discovery, Rockville, MD). We measured serum angiotensin II by enzyme-linked immunosorbent j.jebo.2013.04.005 assay (ELISA) after phenyl extraction (400048, Cayman Chemical, Ann Arbor, MI), as per the manufacturer’s instructions (589301, Cayman).Urine assaysAfter completion of the experimental protocol, urine was collected beginning 2 h prior to organ harvest. jmir.6472 Urine pro-2013 | Vol. 1 | Iss. 6 | e00153 Page?2013 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.M. I. Lygizos et al.Heparanase Mediates Early Septic Renal Dysfunctiontein (#500; Bio-Rad, Hercules, CA) and creatinine (BioAssay Systems, as above) were measured via colorimetric assays. We measured urine HS degradation activity (GWBF49F83; GenWay Biotech, San Diego, CA) as previously described (Schmidt et al. 2012). Urinary IL-6 was measured by ELISA (M6000B; R D Systems, Minneapolis, MN). We measured urinary nitric oxide species (NOx) using a colorimetric assay of urine nitrate (NO3? and nitrite (NO2?, as per the manufacturer’s instructions (780001; Cayman). We m.