Volume 87, Issue 1, Pages 85-94 (January 2015) The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion  Mark C. Dessing, Alessandra Tammaro, Wilco P. Pulskens, Gwendoline J. Teske, Loes M. Butter, Nike Claessen, Marco van Eijk, Tom van der Poll, Thomas Vogl, Johannes Roth, Sandrine Florquin, Jaklien C. Leemans  Kidney International  Volume 87, Issue 1, Pages 85-94 (January 2015) DOI: 10.1038/ki.2014.216 Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 1 S100A8 and S100A9 expression in renal tissue during renal ischemia/reperfusion (I/R) injury. Protein complex S100A8/A9 (a) in kidney tissue from sham (Sh) wild-type (WT) mice or WT mice 1, 5, or 10 days following I/R. Quantification and representative photos from S100A8 (b) or S100A9 (c) single staining in corticomedular region (CM) in kidney tissue slides from WT mice (black bars) and S100A9 knockout (KO) mice (white bars). (d) Representative photos from kidney tissue sections with staining for S100A8 and/or S100A9 or Ly6/S100A8 (original magnification × 400). Protein complex S100A8/A9 expression (a) was corrected for total protein level in tissue. HPF, high-power field. Graph data are mean±s.e.m. *P<0.05, **P<0.005, ***P<0.0005 versus WT sham, #P<0.05, ##P<0.01 versus WT I/R 1. (a): N=8 individuals mice per group, (b, c): N=3 individuals mice per group. Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 2 Renal function and damage in wild-type (WT) and S100A9 knockout (KO) mice during renal ischemia/reperfusion (I/R) injury. Renal function as displayed by serum creatinine (a) and urea (b), renal damage markers as displayed by tubular necrosis score (c, d), Kim1 mRNA (e) and Ngal mRNA (f), apoptotic TECs (g) and proliferating TECs (h) in kidneys from WT (black bars) and S100A9 KO mice (white bars) 1, 5, or 10 days after I/R and in sham (Sh) mice. (d) Representative photos of periodic acid-Schiff diastase (PASD) staining on renal tissue slides from WT and S100A9 KO mice 10 days after I/R. Arrow shows brush border; arrowhead shows cast formation (original magnification × 200). Necrosis score was graded according to a severity score of ‘0’ to ‘5’ as described in Materials and Methods section and displayed as arbitrary units (AU). Amounts of caspase-3- or Ki67-positive TECs were counted in 10 nonoverlapping high-power fields (HPFs). mRNA levels of Kim1 and Ngal (e, f) are expressed relative to housekeeping gene TATA box-binding protein (TBP). Graph data are mean±s.e.m. *P<0.01, **P<0.005, ***P<0.001 versus WT (a–c, e–h). TECs, tubular epithelial cells. Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 3 Renal fibrosis in wild-type (WT) and S100A9 knockout (KO) mice during renal ischemia/reperfusion (I/R) injury. Markers of fibrosis transforming growth factor-beta (TGF-β) (a), hepatocyte growth factor (HGF) (b), collagen type 1 (c, e, and f), and collagen type 3 (d, g, and h) in kidney tissue from WT mice (black bars) and S100A9 KO mice (white bars). TGF-β and HGF expression was corrected for total protein level in tissue. Collagen type 1 and type 3 mRNA levels are expressed relative to housekeeping gene TATA box-binding protein (TBP). Collagen type 1 (e) and type 3 (g) staining on renal tissue slides were digitally analyzed and presented as % positive staining per high-power field (HPF). Representative photos of collagen type 1 (f) and type 3 (h) staining on renal tissue slides from WT and S100A9 KO, 10 days after I/R (original magnification × 200). Graph data are mean±s.e.m. *P<0.05, **P<0.005, ***P<0.001 versus WT. Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 4 Cytokine and chemokine levels in wild-type (WT) and S100A9 knockout (KO) mice during renal ischemia/reperfusion (I/R) injury. Inflammatory mediators keratinocyte-derived chemokine (KC) (a), monocyte chemoattractant protein (MCP)-1 (b), interleukin (IL)-1β (c), and tumor necrosis factor (TNF)-α (d) in kidney homogenate from WT mice (black bars) and S100A9 KO mice (white bars) 1, 5, or 10 days after I/R and in sham (Sh) mice. Protein expression was corrected for total protein level in tissue. Data are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.005 versus WT. Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 5 Macrophage influx in kidneys from wild-type (WT) and S100A9 knockout (KO) mice during renal ischemia/reperfusion (I/R) injury. F4/80 staining on kidney tissue from WT mice (black bars) and S100A9 KO mice (white bars) was digitally analyzed and presented as % positive staining per high-power field (HPF). Representative photos of kidney tissue slides from WT and S100A9 KO mice stained for F4/80, 10 days after I/R (original magnification × 200). Graph data are mean±s.e.m. *P<0.005 versus WT. Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 6 Classical and alternative macrophage markers in ischemia/reperfusion (I/R)-damaged kidney from wild-type (WT) and S100A9 knockout (KO) mice. mRNA levels of general macrophage marker Emr1 (a), classical macrophage marker nitric oxide synthase-2 (NOS-2) (b), interferon-regulating factor-5 (IRF5) (c), and alternative macrophage marker arginase-1 (Arg1) (d), macrophage galactose-type C-type lectin-1 (MGL1) (e), and interferon-regulating factor-4 (IRF4) (f) in kidneys from WT mice (black bars) and S100A9 KO mice (white bars) after I/R or in sham (Sh) mice. mRNA levels are expressed relative to housekeeping gene TATA box-binding protein (TBP). (g) Representative photos for Arg1 single staining on kidney tissue slides from WT and S100A9 KO mice, 5 days after I/R (original magnification × 200). (h) Representative photos for co-localization of Arg1 (red) and F4–80 (blue) on kidney tissue slides from WT and S100A9 KO mice, 5 days after I/R (original magnification × 200). Graph data are mean±s.e.m. *P<0.05, **P<0.005 versus WT. Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 7 S100A8 and S100A9 mRNA levels during macrophage development and polarization. S100A8 and S100A9 mRNA levels in bone marrow cells (BMCs) and bone marrow–derived macrophages (BMDMs) from wild-type (WT) mice (a, b). S100A8 and S100A9 mRNA levels in BMDMs from WT mice, 48h after stimulation with medium (med), lipopolysaccharide+interferon (IFN)-γ (M1 polarization), or interleukin (IL)-4+IL-13 (M2 polarization) (c and d). mRNA levels are expressed relative to housekeeping gene TATA box-binding protein (TBP). Data are mean±s.e.m. (a, b): **P<0.005 versus BMCs, (c, d): *P<0.05, **P<0.005 versus medium (N=6 individuals mice per group). Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 8 Macrophage response to recombinant S100A8 and/or S100A9. Wild-type bone marrow–derived macrophages (WT BMDMs) were cultured in vitro and incubated with medium, 1 or 5μg/ml S100A8 (A8), S100A9 (A9), S100A8/A9 (A8/A9), lipopolysaccharide+interferon (IFN)-γ (M1 polarization), or interleukin (IL)-4+IL-13 (M2 polarization) for 48h. Tumor necrosis factor (TNF)-α was measured in supernatant (a); arginase-1 (Arg1) (b) and interferon-regulating factor 4 (IRF4) (c) were measured on mRNA level. Data are mean±s.e.m. *P<0.05, **P<0.01 versus control (N=3 individuals mice per group). Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 9 In vitro macrophage polarization in wild-type (WT) and S100A9 knockout (KO) mice. Classical macrophage marker nitric oxide synthase-2 (NOS-2) (a, c) and alternative macrophage marker arginase-1 (Arg1) (b, d) in bone marrow–derived macrophages (BMDM; (a, b)) and peritoneal macrophages (PM; (c, d)) from WT mice (black bars) and S100A9 KO mice (white bars) 48h after stimulation with medium (med), lipopolysaccharide+interferon (IFN)-γ (M1 polarization), or interleukin (IL)-4+IL-13 (M2 polarization). mRNA levels are expressed relative to housekeeping gene TATA box-binding protein (TBP). Data are mean±s.e.m. *P<0.05 versus WT (N=6 individuals mice per group). Kidney International 2015 87, 85-94DOI: (10.1038/ki.2014.216) Copyright © 2015 International Society of Nephrology Terms and Conditions