Volume 85, Issue 4, Pages 824-832 (April 2014) Preconditioning with recombinant high-mobility group box 1 protein protects the kidney against ischemia–reperfusion injury in mice  Huiling Wu, Renske Steenstra, Elianne C.S. de Boer, Cathy Y. Zhao, Jin Ma, Jorieke M. van der Stelt, Steven J. Chadban  Kidney International  Volume 85, Issue 4, Pages 824-832 (April 2014) DOI: 10.1038/ki.2013.475 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Recombinant high-mobility group box 1 (rHMGB1)–pretreated mice were protected against renal ischemia–reperfusion injury (IRI). (a) Serum creatinine was significantly lower in rHMGB1-pretreated mice (black bars) as compared with control mice (gray bar) at days 1 and 5 (D1 and D5) after reperfusion. (b, c) Tubular injury was attenuated in rHMGB1-pretreated mice at days 1 and 5 after reperfusion shown on representative sections (hematoxylin and eosin (H&E) stained, original magnification × 200) and semiquantitative analysis. Data shown are mean±s.d.; n=5 per sham group, n=9 per group for day 1, and n=7 per group for day 5. *P<0.05; ***P<0.001. Scale bar=200μm. I/R, ischemia/reperfusion. Kidney International 2014 85, 824-832DOI: (10.1038/ki.2013.475) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Kidney infiltrates were reduced by recombinant high-mobility group box 1 (rHMGB1) preconditioning. (a–d) Neutrophil and macrophage accumulation within the interstitium of the kidney was significantly less in rHMGB1–pretreated mice versus control mice at days 1 and 5 (D1 and D5) after reperfusion by immunohistochemistry staining. Representative sections of kidney stained for (a) neutrophils and (c) macrophages (original magnification × 400) and analysis of (b) neutrophil and (g) macrophage infiltrates in the kidney (numbers/10 high-power fields (HPFs)). (e, h) CD4+ and CD8+ infiltrates were elevated in the kidney at day 5 after IR in control group compared with the sham-operated controls and CD4+ T cells were significantly attenuated in the rHMGB1-pretreated group, but not CD8+ T cells by immunohistochemistry staining. Representative sections of kidney stained for (e) CD4+ and (g) CD8+ (original magnification × 400) and analysis of (f) CD4+and (h) CD8+ infiltrates in the kidney (numbers/10 HPFs). Data shown are mean±s.d.; n=5 per sham group, n=9 per group for day 1, and n=7 per group for day 5. *P<0.05; **P<0.01; ***P<0.001. Scale bar=100μm. I/R, ischemia/reperfusion. Kidney International 2014 85, 824-832DOI: (10.1038/ki.2013.475) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Tubular epithelial cell apoptosis was reduced in the ischemia-reperfusion injury (IRI) kidney by high-mobility group box 1 (HMGB1) preconditioning. (a) Recombinant high-mobility group box 1 (rHMGB1) preconditioning reduced tubular epithelial cell apoptosis in the kidney after ischemia–reperfusion injury (IRI) measured by terminal transferase dUTP nick end labeling (TUNEL) assay on representative sections of kidney. (b) The number of apoptotic tubular cells was significantly lower in rHMGB1-pretreated mice at day 1 after IRI than in control mice. Data shown are as mean±s.d.; n=5 per sham group, n=9 per group for day 1. ***P<0.001. Scale bar=100μm. I/R, ischemia/reperfusion. Kidney International 2014 85, 824-832DOI: (10.1038/ki.2013.475) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Recombinant high-mobility group box 1 (rHMGB1) preconditioning inhibited mRNA upregulation of downstream proinflammatory cytokines and chemokines in kidney ischemia–reperfusion injury (IRI) on day 5 (D5) after reperfusion. Real-time PCR demonstrated that mRNA expression of (a) tumor necrosis factor-α (TNF-α), (b) interleukin-6 (IL-6), (c) CXCL2 (chemokine (C-X-C motif) ligand 2), and (d) CCL2 (chemokine (C-C motif) ligand 2) in the kidney was significantly reduced in rHMGB1-preconditioned (black bar) mice as compared with controls (gray bar). The results have been normalized by expressing the number of transcript copies as a ratio to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data shown are mean±s.d.; n=5 per sham group, n=9 per group for day 1, n=7 per group for day 5. *P<0.05; **P<0.01; ***P<0.001. I/R, ischemia/reperfusion. Kidney International 2014 85, 824-832DOI: (10.1038/ki.2013.475) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 The protective effects of recombinant high-mobility group box 1 (rHMGB1) preconditioning against kidney ischemia–reperfusion injury (IRI) involved Toll-like receptor 4 (TLR4) signaling. (a, b) TLR4 and TLR2 mRNA levels were significantly increased at day 1 and 5 (D1 and D5) after ischemia in WT mice (P<0.01). The increase in TLR4 was significantly reduced by preconditioning at both days 1 and 5 after IRI compared with the controls, whereas the increase in TLR2 was reduced by preconditioning at day 5 only after IRI. TLR4−/− and TLR2−/− mice treated with phosphate-buffered saline (PBS) were protected against (c, d) renal dysfunction and (e, f) tubular damage with additional protection afforded by rHMGB1 preconditioning in TLR2−/− mice but not in TLR4−/− mice. Data shown are mean±s.d.; n=5 per sham group, n=9 per C57BL/6 wild-type (WT) group, n=8 BALB/c WT group, and n=7 per TLR4−/− and TLR2−/− groups. *P<0.05; **P<0.01; ***P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; I/R, ischemia/reperfusion. Kidney International 2014 85, 824-832DOI: (10.1038/ki.2013.475) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 The protective effect of recombinant high-mobility group box 1 (rHMGB1) preconditioning on wild-type (WT) kidney involved sialic acid–binding immunoglobulin (Ig)-like lectin G (Siglec-G) and nuclear factor-κB (NF-κB) activation. (a, b) Interleukin-1 receptor-associated kinase M (IRAK-M) protein was downregulated in ischemia–reperfusion injury (IRI) kidneys 24h after ischemia/reperfusion (I/R) measured by (a) western blot and (b) quantitation of IRAK-M levels by densitometry, normalized to the corresponding β-actin level, n=5 per sham group, n=6 per I/R group. The first six consecutive samples per I/R group were examined because of the size limit of our western blot apparatus. Ctrl, control group. (c, d) mRNA expression of single Ig interleukin-1-related receptor (SIGIRR) and Toll-interacting protein (Tollip) in the kidney was significantly downregulated at day 1 in IRI controls and this was not affected by rHMGB1 preconditioning, n=5 per sham group, n=9 per I/R group. (e) mRNA expression of Siglec-G in IRI kidney in the control group was significantly lower than sham-operated controls at day 1 after operation. In contrast, Siglec-G expression was significantly higher in rHMGB1-preconditioned kidney at day 1 after IR compared with the control kidney, n=5 per sham group, n=9 per I/R group. (f, g) NF-κB DNA-binding activity by electrophoretic mobility shift assay (EMSA) was upregulated in control kidney at day 1 after I/R but this was attenuated in rHMGB1-preconditioned kidney. (f) The assay presented is representative of three experiments. (g) The data include all three experiments. *P<0.05; **P<0.01; ***P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Kidney International 2014 85, 824-832DOI: (10.1038/ki.2013.475) Copyright © 2014 International Society of Nephrology Terms and Conditions