Volume 91, Issue 1, Pages 96-105 (January 2017) Microvesicle transfer of kinin B1-receptors is a novel inflammatory mechanism in vasculitis  Robin Kahn, Maria Mossberg, Anne-lie Ståhl, Karl Johansson, Ingrid Lopatko Lindman, Caroline Heijl, Mårten Segelmark, Matthias Mörgelin, L.M. Fredrik Leeb-Lundberg, Diana Karpman  Kidney International  Volume 91, Issue 1, Pages 96-105 (January 2017) DOI: 10.1016/j.kint.2016.09.023 Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 1 Docking of neutrophil-derived microvesicles expressing B1-receptors to glomerular endothelial cells in kidney biopsies from patients with vasculitis. Kidney biopsies (n = 2) from patients with ANCA-associated vasculitis were labeled with anti-B1-receptor antibody conjugated to 5-nm gold particles and anti-CD66 conjugated to 10-nm gold particles and examined by electron microscopy. (a) Overview of a glomerular capillary (patient 10) showing a glomerular endothelial cell (GEC), the glomerular basement membrane (GBM), podocytes (P), and red blood cells (RBC) within a capillary. The inner box is magnified in B. Bar = 5 μm. (b) Docking of a neutrophil-derived MV to a glomerular endothelial cell. B1-receptors (arrow) were visualized on both MVs and glomerular endothelial membranes. The MVs were neutrophil-derived, i.e., CD66c positive (arrowheads). Bar = 500 nm. (c) Overview of a glomerular capillary (patient 11). Inner box is magnified in D. Bar = 5 μm. (d) Docking of neutrophil-derived MVs to a glomerular endothelial cell. B1-receptors (arrow) present on both MVs and the glomerular endothelial membrane. Glomerular endothelial cells possess native B1-receptor expression. CD66 labeling of the MV is marked with an arrowhead. Bar = 500 nm. (e) Overview of a glomerular capillary (patient 10) labeled with the control antibodies. Inner box is magnified in (f). Bar = 5 μm. (f) MVs on a glomerular endothelial cell. No staining was visualized. Bar = 500 nm. Kidney International 2017 91, 96-105DOI: (10.1016/j.kint.2016.09.023) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 2 Transfer of functional B1-receptors by MVs to HEK cells. MVs generated in HEKB1R cells were incubated with HEKWT cells stimulated with the B1-receptor agonist des-arg10-kallidin. (a) Docking of a MV carrying B1-receptors (arrow, labeled with rabbit anti-human B1-receptor antibody) and transfer of receptors (arrowheads) to the HEKWT cell. (b) Docking of a MV bearing B1-receptors from HEKB1R cells and transfer (arrows) of B1-receptors to a HEKWT cell demonstrated using mouse anti-FLAG M1 antibody, in the presence of des-arg10-kallidin. (c) Labeling for the B1-receptor on HEKWT cells exposed to MVs derived from HEKB1R cells was not seen using conjugated rabbit IgG as the control antibody. (d) Incubation of HEKWT cells with MVs derived from HEKWT cells did not exhibit B1-receptor labeling. (e) Calcium influx into cells. HEKB1R MVs: HEKWT cells incubated with MVs generated in HEKB1R, treated with des-arg10-kallidin, exhibited calcium influx in a median of 50% of cells. A representative image from the experiment is shown on the right, top panel, depicting viable cells in red and calcium influx in green. Double staining (yellow) indicates activated cells. HEKB1R MVs + antagonist: HEKWT cells incubated with MVs generated in HEKB1R and pretreated with the B1-receptor antagonist R715, followed by the agonist showed marked reduction of response to stimulation. A representative image is shown on the right, middle panel. HEKWT MVs: HEKWT cells incubated with MVs generated in HEKWT (lacking B1-receptors), subsequently treated with des-arg10-kallidin, exhibited negligible signaling. A representative image is shown on the right, bottom panel. Magnification of the color images ×40. (f) As a positive control HEKB1R cells treated with des-arg10-kallidin, in the absence of added MVs, exhibited calcium influx in a median of 93% of cells. B1R, B1-receptor. ∗∗P < 0.01, ∗P < 0.05. Kidney International 2017 91, 96-105DOI: (10.1016/j.kint.2016.09.023) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 3 Transfer of functional B1-receptors by MVs from HEKB1R to PGECs or from neutrophils to HEKWT cells. (a) Docking of a MV from HEKB1R (arrows) to a PGEC stimulated with des-arg10-kallidin. B1-receptor expression was assessed using the mouse anti-FLAG M1 antibody showing transfer of receptors from MVs to PGEC. (b) In PGECs incubated with MVs from HEKB1R (MV not visible in the figure), in the absence of the B1-receptor agonist, the FLAG-tagged B1-receptor was present in the intracellular compartment (arrowheads) and only minute staining was seen on the cell surface (arrow). (c) Docking of an MV from HEKB1R (arrow) to a PGEC that expressed endogenous B1-receptors. B1-receptor expression was demonstrated using rabbit anti-human B1-receptor antibody. (d) Minimal endogenous B1-receptor expression (arrow) in a PGEC. (e) Calcium influx into PGEC cells. HEKB1R MVs: PGEC cells, pretreated with TNF-α, incubated with MVs shed from HEKB1R, treated with the agonist des-arg10-kallidin, exhibited calcium influx in a median of 34% of cells. HEKB1R MVs + antagonist: PGEC cells, pretreated with TNF-α, incubated with MVs generated in HEKB1R and pretreated with the B1-receptor antagonist R715, followed by the agonist showed marked reduction of the response to stimulation. HEKWT MVs: PGEC cells, pretreated with TNF-α, incubated with MVs generated in HEKWT (lacking B1-receptors), subsequently treated with the agonist exhibited a low signal. No MVs: PGEC cells, pretreated with TNF-α, treated with des-arg10-kallidin, exhibited a minor calcium influx. (f) Docking of a neutrophil-derived MV expressing B1-receptors (arrows) to a PGEC. (g) Docking (arrow) of a neutrophil-derived MV and transfer of B1-receptors (arrowheads) to a HEKWT cell (these cells do not express endogenous B1-receptors). (h) Calcium influx into HEKWT cells. Neutrophil MVs: HEKWT incubated with MVs shed from neutrophils, treated with the agonist des-arg10-kallidin, exhibited calcium influx in a median of 29% of cells. Neutrophil MVs + antagonist: HEKWT cells incubated with MVs generated in neutrophils and pretreated with the B1-receptor antagonist R715, followed by the agonist, showed marked reduction of the response to stimulation (median 11%). Bar = 500 nm. ∗∗∗P < 0.001, ∗∗P < 0.01,∗P < 0.05, ns, not significant. Kidney International 2017 91, 96-105DOI: (10.1016/j.kint.2016.09.023) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S1 Size and B1 receptor expression on microvesicles. Kidney International 2017 91, 96-105DOI: (10.1016/j.kint.2016.09.023) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S2 Concentration and size distribution of MVs. Kidney International 2017 91, 96-105DOI: (10.1016/j.kint.2016.09.023) Copyright © 2016 International Society of Nephrology Terms and Conditions