Easured urine angiotensin II by ELISA, as per the manufacturer’s AZD-8055 msds instructions (589301; Cayman).positivity. Intensity was defined as the number of pixels positive/image multiplied by average pixel intensity. We performed immunohistochemistry as previously described (Yoshida et al. 2010), using a primary antibody (3G10, 1:100; US Biological, Marblehead, MA) against neoepitopes exposed during HS degradation by heparinase-III (heparitinase), a bacterial analog of mammalian heparanase (Kato et al. 1998; Dull et al. 2012; Schmidt et al. 2012). We performed fluorometric terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (DeadEnd, G3250; Promega, Madison, WI) according to the manufacturer’s instructions.Glomerular filtration rate measurementGlomerular filtration rate (GFR) was measured by inulin clearance, as previously described (Lorenz and Gruenstein 1999). Four hours after CLP or sham surgery, we anesthetized mice with intraperitoneal pentobarbital (60 lg/g body weight) and placed a jugular central venous catheter (PE-10). We infused 0.75 FITC-inulin in 2.25 BSA (in saline) at a rate of 0.5 lL/g body weight/min. After 1 h of infusion, two 30-min collections of urine were obtained via a bladder catheter and weighed for volume determination. Blood for plasma inulin determination was drawn between urine collections. FITC in plasma and urine samples was measured using a CytoFluor plate reader (BioTek Instruments, Winooski, VT). GFR was defined as [inulinurine] 9 (urine output per 30 min)/ [inulinplasma].Assessment of renal vascular permeabilityWe dissolved 0.5 EBD in 4 BSA (in saline). Four hours after CLP, mice were anesthetized with intraperitoneal pentobarbital (60 lg/g body weight) and 20 lg/g body weight EBD-albumin was injected into the right external jugular vein, as previously described (Schmidt et al. 2008). One hour later, we performed a midline laparotomy, exposing the abdominal aorta and kidneys. We killed the anesthetized mice via rapid exsanguination and harvested the left kidney for wet/dry ratio measurement (Schmidt et al. 2008). After flushing the right renal vasculature via arterial injection of saline, we snap-froze the right kidney in liquid nitrogen. We later homogenized the right kidney in 1 mL phosphate buffered saline and digested for 18 h in 2 mL formamide at 60 . We centrifuged the digests at 5000g for 30 min and measured EBD content (in comparison to a standard curve) using spectrophotometry at 620 nm wavelength (Schmidt et al. 2008).HistologyOrgans were formalin-fixed, paraffin-embedded, and sectioned (4 lm) as previously described (Schmidt et al. 2012). An automated tissue stainer (Shandon Varistain Gemini ES, Thermo Scientific, Waltham, MA) performed hematoxylin and eosin (H E) staining. We performed buy Anlotinib immunofluorescence for heparanase (1:1000, Ins-26-2; ProSpec, East Brunswick, NJ) and neutrophil Ly-6B.2 (1:300, clone MCA771G; AbD Serotec, Raleigh, NC) as previously described (Schmidt et al. 2012). As a positive control for Ly-6B.2 immunofluorescence, we harvested lungs from mice 2 h after treatment with 20 lg/g body weight intravenous lipopolysaccharide (LPS, E. coli 055:B5, L2880, Sigma) or 200 lL saline. Ten random images/slide were captured at 1 lm steps (409 objective, 1.4 numerical aperture), and Z-stack SART.S23503 reconstructions was performed using Nikon Elements (Nikon, Melville, NY) (Yoshida et al. 2010). After images were randomized and blinded, we performed image analysis and quantific.Easured urine angiotensin II by ELISA, as per the manufacturer’s instructions (589301; Cayman).positivity. Intensity was defined as the number of pixels positive/image multiplied by average pixel intensity. We performed immunohistochemistry as previously described (Yoshida et al. 2010), using a primary antibody (3G10, 1:100; US Biological, Marblehead, MA) against neoepitopes exposed during HS degradation by heparinase-III (heparitinase), a bacterial analog of mammalian heparanase (Kato et al. 1998; Dull et al. 2012; Schmidt et al. 2012). We performed fluorometric terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (DeadEnd, G3250; Promega, Madison, WI) according to the manufacturer’s instructions.Glomerular filtration rate measurementGlomerular filtration rate (GFR) was measured by inulin clearance, as previously described (Lorenz and Gruenstein 1999). Four hours after CLP or sham surgery, we anesthetized mice with intraperitoneal pentobarbital (60 lg/g body weight) and placed a jugular central venous catheter (PE-10). We infused 0.75 FITC-inulin in 2.25 BSA (in saline) at a rate of 0.5 lL/g body weight/min. After 1 h of infusion, two 30-min collections of urine were obtained via a bladder catheter and weighed for volume determination. Blood for plasma inulin determination was drawn between urine collections. FITC in plasma and urine samples was measured using a CytoFluor plate reader (BioTek Instruments, Winooski, VT). GFR was defined as [inulinurine] 9 (urine output per 30 min)/ [inulinplasma].Assessment of renal vascular permeabilityWe dissolved 0.5 EBD in 4 BSA (in saline). Four hours after CLP, mice were anesthetized with intraperitoneal pentobarbital (60 lg/g body weight) and 20 lg/g body weight EBD-albumin was injected into the right external jugular vein, as previously described (Schmidt et al. 2008). One hour later, we performed a midline laparotomy, exposing the abdominal aorta and kidneys. We killed the anesthetized mice via rapid exsanguination and harvested the left kidney for wet/dry ratio measurement (Schmidt et al. 2008). After flushing the right renal vasculature via arterial injection of saline, we snap-froze the right kidney in liquid nitrogen. We later homogenized the right kidney in 1 mL phosphate buffered saline and digested for 18 h in 2 mL formamide at 60 . We centrifuged the digests at 5000g for 30 min and measured EBD content (in comparison to a standard curve) using spectrophotometry at 620 nm wavelength (Schmidt et al. 2008).HistologyOrgans were formalin-fixed, paraffin-embedded, and sectioned (4 lm) as previously described (Schmidt et al. 2012). An automated tissue stainer (Shandon Varistain Gemini ES, Thermo Scientific, Waltham, MA) performed hematoxylin and eosin (H E) staining. We performed immunofluorescence for heparanase (1:1000, Ins-26-2; ProSpec, East Brunswick, NJ) and neutrophil Ly-6B.2 (1:300, clone MCA771G; AbD Serotec, Raleigh, NC) as previously described (Schmidt et al. 2012). As a positive control for Ly-6B.2 immunofluorescence, we harvested lungs from mice 2 h after treatment with 20 lg/g body weight intravenous lipopolysaccharide (LPS, E. coli 055:B5, L2880, Sigma) or 200 lL saline. Ten random images/slide were captured at 1 lm steps (409 objective, 1.4 numerical aperture), and Z-stack SART.S23503 reconstructions was performed using Nikon Elements (Nikon, Melville, NY) (Yoshida et al. 2010). After images were randomized and blinded, we performed image analysis and quantific.