Volume 76, Issue 11, Pages 1180-1191 (December 2009) COMP-Angiopoietin-1 decreases lipopolysaccharide-induced acute kidney injury  Duk Hoon Kim, Yu Jin Jung, Ae Sin Lee, Sik Lee, Kyung Pyo Kang, Tae Hwan Lee, Sang Yong Lee, Kyu Yun Jang, Woo Sung Moon, Kyu-Sil Choi, Kwon-Ha Yoon, Mi Jeong Sung, Sung Kwang Park, Won Kim  Kidney International  Volume 76, Issue 11, Pages 1180-1191 (December 2009) DOI: 10.1038/ki.2009.387 Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 1 Effects of COMP-Ang1 on mean arterial pressure, renal blood flow, renal vascular resistance, and glomerular filtration rate during endotoxemia in mice. COMP-Ang1 or vehicle was injected 3 days before lipopolysaccharide (LPS, 8 mg/kg, i.p.) or control buffer injection (n=8 in each group). Sixteen hours after LPS or control buffer injection, mean arterial pressure was measured through the carotid artery (a), renal blood flow was measured using a blood flowmeter (b), and renal vascular resistance was calculated as mean arterial pressure/renal blood flow (c). Glomerular filtration rate was measured by inulin clearance (d) (n=10 in each group). COMP-Ang1, COMP-angiopoietin-1; i.p., intraperitoneally; LPS, lipopolysaccharide. **P<0.01 versus control buffer+vehicle; †P<0.05 versus LPS+vehicle. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 2 Effects of COMP-Ang1 on renal endothelial cell injury during endotoxemia in mice. (a–d) Immunofluorescence stain of PECAM-1 in the kidney. Kidneys from mice treated with control buffer plus vehicle (a), control buffer plus COMP-Ang1 (b), lipopolysaccharide (LPS) plus vehicle (c), and LPS plus COMP-Ang1 (d) were harvested 16 h after LPS or control buffer treatment. Scale bar=50 μm and applies to panels a–d. (e) Quantitative score of vWF in kidney. Bar graph shows the area density of the positively stained area to the total field (0.22 μm2). Data are expressed as mean±s.d., n=4 in each group. (f–i) Immunofluorescence double-staining of PECAM-1 and TUNEL in kidney. Scale bar=20 μm and applies to panels f–i. (j) The number of PECAM-1- and TUNEL-positive cells per kidney section (mean area of kidney section; 17.81±2.37 mm2). **P<0.01 versus control buffer+vehicle; †P<0.05 versus LPS+vehicle. (k–m) Electron micrographs of peritubular endothelial and tubular epithelial cells in mice treated with control buffer plus vehicle (k), LPS (8 mg/kg, i.p.) plus vehicle (l), and LPS plus COMP-Ang1 (m). Photographs show representative examples from three experiments per treatment group. Arrows indicate peritubular endothelial cells. T, tubular epithelial cells; RBC, red blood cell. Scale bar=1 μm and applies to panels k–m. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 3 Effects of COMP-Ang1 on the protein expressions of ICAM-1 and VCAM-1 in endotoxemic mice. (a and b) Immunoblot analyses of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in kidney. COMP-Ang1 or vehicle was injected 3 days before lipopolysaccharide (LPS) (8 mg/kg, i.p.) or control buffer injection. Kidneys were harvested 16 h after LPS or control buffer injection. Results from from independent experiments were similar. Densitometric analyses are presented as the relative ratio of ICAM-1 or VCAM-1 to actin. The relative ratio measured in the control buffer plus vehicle group is arbitrarily presented as 1. Data are expressed as mean±s.d. (c–g) Immunohistochemical study of ICAM-1 in kidney. Kidneys from mice treated with control buffer plus vehicle (c), control buffer plus COMP-Ang1 (d), LPS plus vehicle (e), and LPS plus COMP-Ang1 (f) were harvested 16 h after LPS or control buffer treatment. Scale bar=50 μm. (g) Quantitative score of ICAM-1 in kidney (n=4 in each group). Bar graph shows the area density of the positively stained area to the total field (0.22 μm2). Data are expressed as mean±s.d. **P<0.01 versus control buffer+vehicle; †P<0.05 versus LPS+vehicle. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 4 Immunofluorescence study of ER-HR3-positive macrophages in kidney. Kidneys from mice treated with control buffer plus vehicle (a–c), control buffer plus COMP-Ang1 (d–f), lipopolysaccharide (LPS) (5 mg/kg, i.p.) plus vehicle (g–i), and LPS plus COMP-Ang1 (j–l) were harvested 5 days after LPS or control buffer treatment. Tissues were fixed in 4% formaldehyde solution and frozen sections were then stained with ER-HR3 antibody (a, d, g, and j) and DAPI (b, e, h, and k). Scale bar=50 μm. Quantitative score of ER-HR3-positive macrophages (m) in kidney, n=5 for each experimental group. Bar graph shows the number of the unit area. Data are expressed as mean±s.d. **P<0.01 versus control buffer+vehicle; †P<0.05 versus LPS+vehicle. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 5 Serum nitric oxide (NO) levels and renal inducible nitric oxide synthase (iNOS) protein expression during endotoxemic acute kidney injury. (a) Serum NO levels were measured using a nitrate/nitrite colorimetric assay. Serum was collected at 16 h after intraperitoneal injection of lipopolysaccharide (LPS) (8 mg/kg) (n=4 in each group) (b) Immunoblotting was used to examine iNOS protein expression in the kidney from mice treated with control buffer plus vehicle, control buffer plus COMP-Ang1, LPS plus vehicle, and LPS plus COMP-Ang1. Kidneys were harvested at 16 h after LPS administration. The immunoblot was probed with an anti-iNOS antibody and reprobed with an anti-actin antibody to control for protein loading in each lane. Densitometric analyses are presented as the relative ratio of each protein to actin. The relative ratio measured in kidneys from mice treated with control buffer plus vehicle is arbitrarily presented as 1. Bars represent the mean±s.d. from four experiments. **P<0.01 versus control buffer+vehicle; ‡P<0.01 versus LPS+vehicle. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 6 Renal vascular permeability and renal tissue blood flow in the kidneys of lipopolysaccharide (LPS)-induced endotoxemic mice. LPS, control buffer, COMP-Ang1, and vehicle were administered as described in the Materials and Methods section. (a) Renal vascular permeability was quantified with Evans blue dye, n=6 for each experimental group. (b) Renal cortical and medullary blood flow was measured with laser-Doppler ultrasonography, n=5 for each experimental group. Data are expressed as means±s.d. **P<0.01 versus control buffer+vehicle; †P<0.05 versus LPS+vehicle. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 7 Confocal laser scanning microscopy of rhodamine in the kidneys of lipopolysaccharide (LPS)-induced endotoxemic mice. The oxidation of dihydrorhodamine-123 (DHR) to rhodamine was used as an indicator of reactive oxygen species/reactive nitrogen species generation in kidney. COMP-Ang1 or vehicle was administered 3 days before the injection of LPS (8 mg/kg, i.p.) or control buffer. DHR (3 mg/kg) was administered i.v. 16 h after the injection of LPS. Kidneys from mice treated with control buffer plus vehicle (a–c), control buffer plus COMP-Ang1 (d–f), LPS plus vehicle (g–i), and LPS plus COMP-Ang1 (j–l) were harvested 15 min after the DHR injection. Note that the intensity of rhodamine was increased in renal tubular epithelial cells (g; arrow and inset), and this increased intensity was lower in the LPS plus COMP-Ang1 group than in the LPS plus vehicle group, n=3 for each experimental group. Scale bar=50 μm. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 8 Intravital microscopic images of rhodamine fluorescence from the kidney from lipopolysaccharide (LPS)-induced endotoxemic mice. The oxidation of dihydrorhodamine-123 (DHR) to rhodamine was used as an indicator of reactive oxygen species/reactive nitrogen species generation in kidney. LPS, COMP-Ang1, and DHR were administered as described in Figure 7 legend. Kidneys from mice treated with control buffer plus vehicle (a), control buffer plus COMP-Ang1 (b), LPS plus vehicle (c), and LPS plus COMP-Ang1 (d) were observed 15 min after the DHR injection. Note that the expression of rhodamine in renal tubular epithelial cells from mice treated with LPS plus vehicle was lower in kidneys from mice treated with LPS plus COMP-Ang1, n=3 for each experimental group. Scale bar=50 μm. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 9 Immunoblot analyses of Akt and phospho-Akt from the kidneys of lipopolysaccharide (LPS)-induced endotoxemic mice. Kidneys were harvested 16 h after LPS or control buffer injection. Mice were pretreated with 1 × 108 PFU Ade-sTie2-Fc 24 h before treatment with 1 × 108 PFU Ade-COMP-Ang1. COMP-Ang1 adenovirus was administered 3 days before LPS injection. Each lane contains 150 μg total protein. Blots were probed with an anti-phospho-Akt (p-Akt) antibody. The membrane was stripped and reprobed with an anti-Akt antibody to control for protein loading in each lane. Results were similar from six independent experiments. Densitometric analyses are presented as the relative ratio of p-Akt to Akt. The relative ratio measured in kidneys treated with control buffer plus vehicle is arbitrarily presented as 1. Results from three independent experiments were similar. Data are expressed as mean±s.d. *P<0.05 versus control buffer+vehicle; #P<0.05 versus control buffer+vehicle. Kidney International 2009 76, 1180-1191DOI: (10.1038/ki.2009.387) Copyright © 2009 International Society of Nephrology Terms and Conditions