Volume 88, Issue 5, Pages 1030-1046 (November 2015) Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)- associated glomerulonephritis  Kim M. O’Sullivan, Camden Y. Lo, Shaun A. Summers, Kirstin D. Elgass, Paul J. McMillan, Anthony Longano, Sharon L. Ford, Poh-Yi Gan, Peter G. Kerr, A. Richard Kitching, Stephen R. Holdsworth  Kidney International  Volume 88, Issue 5, Pages 1030-1046 (November 2015) DOI: 10.1038/ki.2015.202 Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 1 | Assessment of leukocytes and fibrin in MPO-ANCA GN biopsies. (a–c) Frequencies of macrophages, neutrophils, and T cells were assessed by immunostaining in biopsies of MPO-ANCA GN patients (n=47) and controls (minimal change and thin basement membrane disease, n=10), shown as the mean cells per glomerular cross-section (C/GCS, a, b) or cells per high-powered field (C/HPF, c). (a) MPO-ANCA GN patients had significantly more glomerular macrophages (Mϕ), neutrophils (PMNs), and CD4+ and CD8+ cells than those of the control cohort. (b) The predominant periglomerular leukocyte in the MPO-ANCA GN cohort were macrophages, followed by T cells, with CD4+ and CD8+ cells being present in equal numbers and then neutrophils. (c) Interstitial T cells were the predominant cell type within the interstitium, with CD4+ and CD8+ cells being detected in similar numbers. (d–h) Correlation of leukocytes with eGFR demonstrated that (d) interstitial CD4+ T cells, (e) CD8+ T cells, (f) macrophages, and (g) neutrophils were correlated with a low eGFR at biopsy. (i) MPO-ANCA GN patients had significantly more fibrin deposition in glomerular lesions than control patients. ANCA, antineutrophil cytoplasmic antibody;eGFR estimated glomerular filtration rate;GN, glomerulonephritis;MPO, myeloperoxidase;PMN, polymorphonuclear leukocyte. **P<0.005, ***P<0.0005, ****P<0.0001. Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 2 Extracellular MPO is detected in glomeruli and the interstitium of MPO-ANCA GN patients. An anti-CD45 antibody (a panleukocyte marker, shown in green) and an anti-MPO antibody (shown in red) were used to define the expression of intracellular and extracellular MPO in MPO-ANCA GN kidneys. Nuclei were stained blue with DAPI. (a) Low-powered image (×600) reveals MPO within CD45+ leukocytes (yellow in the merged image) and extracellular (red in the merged image) in a mildly affected glomerulus. (b) High-powered images (yellow bordered inset from a) demonstrating intracellular (yellow, arrowheads) and extracellular MPO (red, arrows). (c) Extracellular MPO (red) in a segmentally damaged glomerulus (segmental lesion with loss of DAPI+ cells) with few CD45+ cells (green, large arrowhead) is shown. (d) High- powered images (yellow bordered inset from c) showing multiple deposits of extracellular MPO (arrows), with several MPO+CD45+ cells (one indicated by arrowhead). (e) High-powered images of the tubulointerstitium demonstrating extracellular MPO (arrows) and CD45+MPO+ cells. Large-scale bars=50μm, small-scale bars=10μm. ANCA, antineutrophil cytoplasmic antibody;DAPI, 4′,6-diamidino-2-phenylindole;GN, glomerulonephritis;MPO, myeloperoxidase. Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 3 Substantial amounts of MPO are deposited in kidneys of patients with MPO-ANCA GN. (a) Image analysis demonstrates that intracellular MPO and deposited extracellular (‘extraleukocyte’) MPO are higher in glomeruli of MPO-ANCA GN patients (n=47, shown as arbitrary units per glomerular cross-section (AU/GCS)) than that of control disease group where extracellular MPO is virtually absent (n=10). (b) A similar result is shown for the tubulointerstitial/periglomerular regions where significant amounts of extracellular MPO were deposited per high-powered field (HPF). The proportion (expressed as a percentage) of MPO that was deposited (extracellularly) within glomeruli (c) and the tubulointerstitial/periglomerular regions (d) was higher than that of the control disease group. (e) Extracellular MPO was deposited in a similar manner in PR3-ANCA GN patients (n=11). (f) Serial sections of MPO-ANCA GN patients (n=10) were stained for CD15 (neutrophils) and MPO and CD68 (macrophages). CD15+ neutrophils were associated with a greater proportion of deposited MPO than CD68+ macrophages. ANCA, antineutrophil cytoplasmic antibody;GN, glomerulonephritis;MPO, myeloperoxidase;PR3, proteinase 3. ***P<0.0005, ****P<0.0001. Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 4 | Numbers and proportions of MPO-expressing neutrophils and macrophages. (a) Kidney sections were double labeled for MPO (red) and CD15 (neutrophils (PMN), green). All neutrophils were MPO+ (yellow, arrow). (b) Kidney sections labeled for MPO (red) and CD68 (macrophage/monocytes (Mϕ), green) showed that approximately one-third of all macrophages were MPO+. (c–e) The frequencies of MPO+ intrarenal neutrophils and macrophages were significantly higher in the MPO-ANCA GN group (n=48) than those of the control disease group (n=10). All neutrophils and a proportion of macrophages were MPO+, with each cell type contributing approximately one-half of MPO+ leukocytes. Patterns in the glomerular, periglomerular, and interstitial areas were similar. Scale bars in insets =10μm, large pictures=50pm. ANCA, antineutrophil cytoplasmic antibody;DAPI, 4',6-diamidino-2-phenylindole;GN, glomerulonephritis;MPO, myeloperoxidase;PMN, polymorphonuclear leukocyte. **P<0.005, ****P=0.0001. Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 5 Neutrophil extracellular traps (NETs) are found in the glomeruli of patients with MPO-ANCA GN. (a) Extracellular traps were identified as the colocalization of MPO (red), H2A-H2B histones (green), and chromatin (DAPI, blue). (b) High-magnification image of yellow inset from a (scale bar=5μm). To demonstrate that neutrophils produce extracellular traps within glomeruli, (c) sections were stained for H2A-H2B (histones, green), DAPI (chromatin, blue), and MPO (red) and compared with (d) serial sections stained for H2A-H2B, DAPI, and neutrophil elastase (red, neutrophils). NETs were identified by colocalization of all three markers (white) in both sections (scale bar=50μm). (e, f) High-powered images of inset areas of c and d demonstrate neutrophils producing NETs (arrows) and some extracellular trap-producing cells that are negative for neutrophil elastase (arrowhead). Scale bar =10μm. ANCA, antineutrophil cytoplasmic antibody;DAPI, 4′,6-diamidino-2- phenylindole;GN, glomerulonephritis;MPO, myeloperoxidase Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 6 | Quantitation of glomerular NETs by serial sections and their association with extracellular MPO and neutrophils. (a) Of the 27 (of 44) patients in which intraglomerular NETs were detected, >59% demonstrated NETs in over half their glomeruli (by analysis of serial sections probed for DAPI, H2A-H2B, and either MPO or neutrophil elastase). (b) Intraglomerular neutrophil (PMN) numbers were significantly higher in patients with NETs compared with those of glomeruli with no NETs. (c) Quantitative image analysis demonstrates significantly higher amounts of extracellular MPO in NET-positive patients. (d) Compared with patients who had no NETs in glomeruli, those with intraglomerular NETs showed increased total glomerular MPO. AU, arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindole;GCS, glomerular cross-section; MPO, myeloperoxidase; NET, neutrophil extracellular trap; PMN, polymorphonuclear leukocyte.*P<0.05. Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 7 Serial sections of glomerular NETs are positive for PAD4 and H3Cit. (a) Ten serial sections from patients with NETs within glomeruli identified with H2A-H2B, MPO, and DAPI demonstrate the percentage of glomeruli with NETs is identical by staining with the NET constituents MPO, H3Cit, and PAD4. (b) Sections stained for H2A-H2B (histones, green), DAPI (chromatin, blue), and MPO (red) and compared with (c) serial sections stained for H3Cit (green) MPO (red), and PAD4 (blue);arrows indicate areas of NET production within glomeruli (scale bar=50μm). NETs were identified by colocalization of all three markers in each serial section. In the high-magnification cellular inset, arrows indicate the same cell in each serial section (scale bar=5μm). H3Cit, citrullinated histone H3;DAPI, 4',6-diamidino-2-phenylindole; MPO, myeloperoxidase;NET, neutrophil extracellular trap;PAD4, peptidyl arginine 4 deiminase. Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 8 Super-resolution microscopy images of NETs. Three-dimensional structural illumination (3D-SIM) super-resolution microscopy of a further five samples from patients with NETs in glomeruli demonstrates the morphological difference between intact neutrophils, cell debris from an apoptotic or necrotic cell, and NET-forming cells. (a) Intact neutrophil, with MPO (red), PAD4 (blue, dense nuclear localization), and no H3Cit staining (green). (b) Cellular debris from an apoptotic or necrotic MPO+ cell, with MPO (red), minimal PAD4 (blue), H3Cit (green), and no colocalization evident. (c) NET-forming cell or cells in vivo with definitive colocalization of the NET constituents MPO (red), H3Cit (green), and PAD4 (blue) and in a serial section (d) stained (arrows) for MPO (red), H3Cit (green), and H2A-H2B (blue). Cells were also positive for neutrophil elastase by a further serial section (not shown). Lower-powered image magnification, scale bar=50μm. Super-resolution images, scale bar =10μm. H3Cit, citrullinated histone H3;DAPI, 4′,6-diamidino-2-phenylindole;MPO, myeloperoxidase;NET, neutrophil extracellular trap; PAD4, peptidyl arginine 4 deiminase. Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 9 Glomerular MPO+ macrophages produce extracellular trap-like structures (METs). To determine whether neutrophil elastase-negative extracellular traps (that were positive for MPO/histones/DAPI) were derived from MPO+ macrophages, serial sections from 10 MPO- ANCA patients with glomerular extracellular trap formation were probed for (a) MPO, (red)/H2A–H2B histones (green)/DAPI (blue), (b) MPO (red)/ CD68 (green)/DAPI (blue). and (c) neutrophil elastase (red)/H2A–H2B histones (green)/DAPI (blue). In addition to neutrophils producing extracellular traps, MPO+ macrophages in glomeruli also can form extracellular traps. Arrowheads indicate the same cell in three panels being positive for MPO/histones/DAPI and CD68 (macrophage marker), but negative for neutrophil elastase, confirming it is a macrophage extruding extracellular trap-like structures. Low-powered images scale bars=50μm. High-powered images, scale bar =10μm. ANCA, antineutrophil cytoplasmic antibody;DAPI, 4′,6-diamidino-2-phenylindole;MPO, myeloperoxidase;NE, neutrophil elastase. Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 10 | Endothelial cells and podocytes demonstrated positive staining for MPO. Endothelial cells and podocytes shed their identifying cell surface markers CD34 (endothelial cells) and nephrin (podocytes) as glomerular injury becomes more severe. (a) Normal glomerulus from minimal change disease demonstrated intact CD34+ endothelial cells (green) with no intraglomerular MPO (no red staining). Scale bar=50μm. (b) CD34+ intact capillary loops positive for MPO (yellow; inset×1200 scale bar=20μm). (c) Injured glomerulus with less CD34+ endothelial cells staining (green) and with MPO+ areas (red). Scale bar=50μm (inset, scale bar=20pm). (d) More severe glomerular injury with no CD34+ endothelial cells, but capillaries lined with MPO deposits (inset scale bar=20μm). (e) To further characterize cells positive for MPO, quadruple staining for endothelial cells (CD34, green), podocytes (nephrin, white), MPO (red), and DAPI (blue) was performed. In some glomeruli, there are areas of CD34+ capillary loops still intact that colocalize with MPO (scale bar=50μm). Inset shows MPO is close to capillary walls (arrowhead) and near podocytes (arrow) (scale bar=10μm). DAPI, 4′,6-diamidino-2-phenylindole; MPO, myeloperoxidase Kidney International 2015 88, 1030-1046DOI: (10.1038/ki.2015.202) Copyright © 2015 International Society of Nephrology Terms and Conditions