Volume 83, Issue 5, Pages 865-877 (May 2013) Leukocyte-derived MMP9 is crucial for the recruitment of proinflammatory macrophages in experimental glomerulonephritis  Malte A Kluger, Gunther Zahner, Hans-Joachim Paust, Melanie Schaper, Tim Magnus, Ulf Panzer, Rolf A K Stahl  Kidney International  Volume 83, Issue 5, Pages 865-877 (May 2013) DOI: 10.1038/ki.2012.483 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Renal phenotype of matrix metalloproteinase 9 (MMP9)-/- mice. (a) Representative photographs of periodic acid–Schiff (PAS)-stained kidney sections of 10-week-old wild-type (WT) and MMP9-/- mice (magnification × 400). (b) Blood urea nitrogen (BUN) levels and (c) albumin–creatinine ratio of 10-week-old WT and MMP9-/- mice (n=14 per group). (d) Total body weight and kidney weight, as well as the number of glomeruli per low-power field (e, magnification × 200), of 10-week-old WT and MMP9-/- mice do not differ significantly (n=7 per group). Symbols represent individual data points; horizontal lines indicate mean values. Kidney International 2013 83, 865-877DOI: (10.1038/ki.2012.483) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Attenuated course of nephrotoxic crescentic glomerulonephritis (NTN) in matrix metalloproteinase 9 (MMP9)-/- mice. (a) Representative photographs of periodic acid–Schiff (PAS)-stained kidney sections of wild-type (WT) and MMP9-/- mice at day 10 after induction of NTN or control (magnification × 400). (b) Representative photographs of fibrin-stained kidney sections of WT and MMP9-/- mice at day 10 after induction of NTN or control (magnification × 400). (c) Quantification of glomerular crescents in nephritic WT (n=13) and MMP9-/- mice (n=17), and in non-nephritic controls (n=14 each) at day 10 of NTN. (d) Glomerular fibrin deposition in nephritic WT and MMP9-/- mice, and in non-nephritic controls at days 2 and 10 of NTN was quantified using an arbitrary semiquantitative staining index (0: no staining, 1: light staining, 2: moderate staining, 3: heavy staining). (e) Blood urea nitrogen (BUN) levels (left) and albumin/creatinine ratio (middle) of nephritic WT mice, nephritic MMP9-/- mice, and their corresponding controls at day 10 after NTN or unspecific sheep IgG injection. Total body weight (right) was determined before and after induction of NTN. Symbols represent individual data points; horizontal lines indicate mean values. *P<0.01; **P<0.001; ***P<0.0001. Kidney International 2013 83, 865-877DOI: (10.1038/ki.2012.483) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Renal leukocyte recruitment. (a) Representative photographs of kidney sections from nephritic wild-type (WT) and matrix metalloproteinase 9 (MMP9)-/- mice and non-nephritic controls immunohistochemically stained for the monocyte marker F4/80 and the pan T-cell marker CD3 at day 10 of nephrotoxic crescentic glomerulonephritis (NTN; magnification × 400). (b) Quantification of tubulointerstitial and periglomerular F4/80+ (left) and intraglomerular MAC2+ monocytes (right). (c) Quantification of tubulointerstitial (left) and intraglomerular CD3+ T cells (right) in non-nephritic (n=14) and nephritic WT (n=13) and MMP9-/- mice (n=17) at day 10 after induction of NTN. Symbols represent individual data points; horizontal lines indicate mean values. *P<0.01; **P<0.001; ***P<0.0001. Kidney International 2013 83, 865-877DOI: (10.1038/ki.2012.483) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Characterization of intrarenal monocytes in nephritic matrix metalloproteinase 9 (MMP9)-/- and wild-type (WT) mice. (a) Kidney cells were gated for CD45+ and CD11b+ events (left). CD11b+Ly6G+ neutrophils were quantified (right). (b) Intrarenal dendritic cells (DC) and macrophages (Mφ) were discriminated by CD11c staining. (c) CD11b+ CD11c+ DCs (left), as well as their fraction of MHCII+ (middle) and Ly6C+ cells (right), were quantified. (d) Intrarenal macrophages were analyzed for their expression of the activation markers F4/80 (left), MHCII (middle), and Ly6C (right). The upper rows show representative fluorescence-activated cell sorting (FACS) plots; the bottom rows show percentages of gated cells. (e) Intrarenal CD45+CD11b+Ly6G-CD11c- macrophages were FACS-sorted and analyzed for their mRNA expression. Expression shown as x-fold of non-nephritic controls. *P<0.01; **P<0.001; n=5 per group. Kidney International 2013 83, 865-877DOI: (10.1038/ki.2012.483) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Chemokine and cytokine mRNA expression profile after proinflammatory stimulus. (a) mRNA was isolated from total renal cortex of nephritic and non-nephritic wild-type (WT) or matrix metalloproteinase (MMP)9-/- mice and quantified by quantitative reverse transcriptase (RT)-PCR at day 10 after induction of nephrotoxic crescentic glomerulonephritis (NTN). Levels are expressed as x-fold of genotype-matched non-nephritic controls. No MMP9 mRNA could be found in MMP9-/- mice. (b) Real-time RT-PCR was also performed with mRNA derived from peritonitis-elicited macrophages (PEM) 3h after lipopolysaccharide (LPS) stimulation. Levels are expressed as x-fold of unstimulated cells derived from the same animals cultured in identical but LPS-free medium. *P<0.01; **P<0.001. Kidney International 2013 83, 865-877DOI: (10.1038/ki.2012.483) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Migratory capacity of peritoneal leukocytes from matrix metalloproteinase 9 (MMP9)-/- mice. Peritonitis-elicited macrophages (PEM) were isolated 4 days after intraperitoneal injection of thioglycollate from wild-type (WT) and MMP9-/- mice. Fluorescence-activated cell sorting (FACS) plots were gated on CD45+ events. About 90% of CD45+ cells were CD11b+ and F4/80+. About 3.5% of CD45+ cells were CD4+ cells. Migration of peritoneal macrophages (a) and TH lymphocytes (b) against 100ng/ml chemokine (C-C motif) ligand 2 (CCL2) or C-X-C motif chemokine 10 (CXCL10) was quantified and characterized by FACS analysis in a chemotactic Transwell assay. Chemotactic index was determined as x-fold of spontaneous migration toward chemokine-free medium. **P<0.001. Kidney International 2013 83, 865-877DOI: (10.1038/ki.2012.483) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Attenuated course of nephrotoxic crescentic glomerulonephritis (NTN) after whole-body irradiation and transplantation of matrix metalloproteinase 9 (MMP9)-deficient bone marrow. (a) Representative photographs of periodic acid–Schiff (PAS)-stained kidney sections at day 10 after induction of NTN (magnification × 400, group names: genotype recipient/genotype donor). (b) Representative photographs of kidney sections immunohistochemically stained for the monocyte marker F4/80 at day 10 after induction of NTN (magnification × 400, group names: genotype recipient/genotype donor). (c) Quantification of glomerular crescents (left), blood urea nitrogen levels (middle), and albumin/creatinine ratio (right) of nephritic mice at day 10 after NTN. (d) Quantification of F4/80+ interstitial (left) and MAC2+ glomerular monocytes (right). (e) Tubulointerstitial (left) and glomerular CD3+ T cells (right) at day 10 of NTN. (f) mRNA expression profile: mRNA was isolated from total renal cortex of nephritic mice and quantified by quantitative reverse transcriptase –PCR at day 10 after induction of NTN. Levels are expressed as x-fold of WT/WT mice. No MMP9 mRNA could be detected in MMP9-/-/MMP9-/- mice. *P<0.01; **P<0.001; n=6 per group. Kidney International 2013 83, 865-877DOI: (10.1038/ki.2012.483) Copyright © 2013 International Society of Nephrology Terms and Conditions