1. Volume 86, Issue 2, Pages 316-326 (August 2014)
The high-mobility group protein B1–Toll-like receptor 4 pathway contributes to the acute lung injury induced by bilateral nephrectomy 
Kent Doi, Tomoko Ishizu, Maki Tsukamoto-Sumida, Takahiro Hiruma, Tetsushi Yamashita, Emi Ogasawara, Yoshifumi Hamasaki, Naoki Yahagi, Masaomi Nangaku, Eisei Noiri 
Kidney International 
Volume 86, Issue 2, Pages (August 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Neutrophil infiltration in acute lung injury (ALI) induced by bilateral nephrectomy (BNx). BNx induced remarkable neutrophil infiltration in the lung at 6h after surgery. Neutrophil infiltration was observed using Giemsa staining in (a) nephrectomized and (b) sham-operated C3H/HeN mice (arrows). Original magnification × 400. Bar=50μm. (c) The neutrophil count in the lung was significantly smaller in Toll-like receptor 4 (TLR4)–mutant C3H/HeJ mice than in TLR4-wild-type C3H/HeN mice (n=6 in each group). Pulmonary mRNA expressions of (d) tumor necrosis factor-α (TNF-α), (e) keratinocyte-derived chemokine (KC), and (f) interleukin 6 (IL-6) were increased by BNx, whereas C3H/HeJ mice showed significantly reduced expressions of these inflammatory cytokines (n=6 in each group). *P<0.05 between C3H/HeJ and C3H/HeN, #P<0.05 versus sham.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

3. Figure 2
Neutrophil elastase (NE) and myeloperoxidase (MPO) activity after acute kidney injury (AKI) by bilateral nephrectomy (BNx). (a) The neutrophil count in the lung was significantly higher at 6h after BNx than at 24h in C3H/HeN and C3H/HeJ mice (n=6 in each group). (b) Plasma and (c) lung NE activity and (d) lung MPO activity after BNx were examined. BNx increased plasma and pulmonary NE activity and C3H/HeJ mice showed significantly reduced NE activity at 6h after BNx (n=4 in sham group, n=7 in BNx group). *P<0.05 between C3H/HeJ and C3H/HeN, **P<0.05 between 6h and 24h, #P<0.05 versus sham.
Kidney International  , DOI: ( /ki )
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4. Figure 3
Lipid peroxidation in the lung of bilateral nephrectomized mice. Immunohistochemistry of 4-hydroxy-2-hexenal (HHE) revealed that bilateral nephrectomy (BNx) increased lipid peroxidation in the lung of C3H/HeN mice and signal intensity in C3H/HeJ mice was markedly diminished. Original magnification × 600. Bar=50μm.
Kidney International  , DOI: ( /ki )
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5. Figure 4
Vascular permeability in the lung of bilateral nephrectomized mice. Evans blue dye leakage in the lung was measured at 6 and 24h after bilateral nephrectomy (BNx; n=4 in sham group, n=6 in BNx group). BNx increased pulmonary vascular permeability, and C3H/HeJ mice showed significantly reduced leakage at 6h after BNx. *P<0.05 between C3H/HeJ and C3H/HeN, #P<0.05 versus sham.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
Splenocyte transfer reversed the resistance of C3H/HeJ mice. Injection of splenocyte isolated from Toll-like receptor 4 (TLR4)–wild-type C3H/HeN mice to TLR4-mutant C3H/HeJ mice canceled the resistance against lung injury by bilateral nephrectomy (BNx) evaluated by (a) plasma and (b) lung neutrophil elastase (NE) activity (n=4 in sham group, n=7 in BNx group). #P<0.05 versus sham.
Kidney International  , DOI: ( /ki )
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7. Figure 6
Blood high-mobility group protein B1 (HMGB1) levels were increased equally in C3H/HeJ and C3H/HeN mice. Blood HMGB1 levels were measured at 6h after bilateral nephrectomy (BNx). No significant difference was found between C3H/HeJ and C3H/HeN mice (n=4 in sham group, n=7 in BNx group). #P<0.05 versus sham.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Neutralizing antibody (Ab) against high-mobility group protein B1 (HMGB1) reduced lung injury by bilateral nephrectomy (BNx). (a) Plasma and (b) lung neutrophil elastase (NE) activity after BNx was examined. C3H/HeJ mice showed significantly lower NE activity than C3H/HeN mice at 6h after BNx. Treatment with anti-HMGB1 Ab reduced NE activity only in Toll-like receptor 4 (TLR4)–wild-type C3H/HeN mice. (c) The neutrophil count in the lung showed similar results to those of NE activity. (d) Blood high-mobility group protein B1 (HMGB1) levels were not influenced by anti-HMGB1 Ab treatment (n=7 in IgY group, n=6 in anti-HMGB1 antibody group). #P<0.05 versus sham, *P<0.05 between C3H/HeJ and C3H/HeN, **P<0.05 between IgY and anti-HMGB1 Ab.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

9. Figure 8
Neutralizing antibody (Ab) against high-mobility group protein B1 (HMGB1) reduced keratinocyte-derived chemokine (KC) mRNA expression in acute lung injury (ALI) induced by bilateral nephrectomy (BNx). Increased pulmonary mRNA expressions of (a) tumor necrosis factor-α (TNF-α), (b) KC, and (c) interleukin 6 (IL-6) by BNx were evaluated. C3H/HeJ mice showed significantly lower expression of these inflammatory cytokines compared with C3H/HeN mice (n=4 in sham group, n=6 in BNx group). Administration of anti-HMGB1 Ab significantly reduced KC mRNA expression in C3H/HeN mice. #P<0.05 versus sham, *P<0.05 between C3H/HeJ and C3H/HeN, **P<0.05 between IgY and anti-HMGB1 Ab.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

10. Figure 9
Neutralizing antibody (Ab) against high-mobility group protein B1 (HMGB1) reduced lung injury by renal ischemia reperfusion (IR). (a) Plasma and (b) lung neutrophil elastase (NE) activity and (c) neutrophil infiltration in the lung after renal IR were examined. No difference between C3H/HeN and C3H/HeJ mice was found in NE activity and neutrophil count at 6h after renal IR. Treatment with anti-HMGB1 Ab reduced NE activity not only in C3H/HeN mice (plasma and lung) but also in C3H/HeJ mice (lung). The neutrophil count in the lung showed similar results to those of lung NE activity. (d) Blood HMGB1 levels were not influenced by anti-HMGB1 Ab treatment (n=4 in sham group, n=6 in IgY group, n=6 in anti-HMGB1 antibody group). #P<0.05 versus sham, **P<0.05 between IgY and anti-HMGB1 Ab.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions