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. 2020 Jun;97(6):1196–1207. doi: 10.1016/j.kint.2019.12.014

Figure 1.

Figure 1

Spleen tyrosine kinase (SYK) is expressed and activated at sites of disease in experimental autoimmune vasculitis. Immunohistochemical staining for total (T)-SYK, phosphorylated (P)-SYK, and ED-1 (rat homologue of CD68) in healthy and diseased rat lung and renal tissue 6 weeks after induction of experimental autoimmune vasculitis (EAV). (a,b) T-SYK detection in a healthy lung, demonstrating (a) SYK expression in large airway cuboidal epithelial cells and associated lymphoid tissue, but (b) minimal SYK detection in alveolar squamous epithelium. (c) T-SYK detection in inflamed lung tissue, demonstrating a population of large mononuclear cells that are positive for SYK, with alveolar consolidation by erythrocytes. (d–g) Staining of serial sections of lung tissue showing an alveolar lumen containing mononuclear cells positive for (d) ED-1, (e) T-SYK, and (f) P-SYK. Double staining confirms co-localization of T-SYK (brown) and ED-1 (blue) in these alveolar cells. (h) Glomerular T-SYK detection in adjacent crescentic and normal glomeruli in nephritic kidney tissue, demonstrating SYK detection within proliferative lesions in diseased glomeruli, although no expression in preserved, non-inflamed glomeruli. (i,j) RNAScope (Advanced Cell Diagnostics, Newark, CA) in situ hybridization for SYK mRNA, stained in purple, in (i) nephriritic and (j) normal glomeruli. (k) SYK mRNA expression in rat renal tissue 6 weeks after induction of EAV and in control rats immunized with complete Freunds adjuvant alone, confirming upregulation of SYK expression in nephritic tissue (n = 4 per group; statistical comparison by Mann-Whitney test; ∗P < 0.05). (l) Double staining demonstrating co-localization of ED-1 (blue) and T-SYK (brown) with a segmental area of inflammation in a nephritic glomerulus (solid arrowhead), and a small population of T-SYK+ ED-1 cells (open arrowhead). (m) ED-1 and (n) P-SYK staining in sequential sections from nephritic rat tissue, suggesting co-localization of SYK activation with infiltrating ED-1 expressing monocytes/macrophages. (o) Immunoblotting for T-SYK and P-SYK in whole kidney tissue, confirming upregulation of SYK phosphorylation during EAV compared to complete Freunds adjuvant control animals. (Representative photomicrographs, original magnification [a] ×100, [b] ×200, and [c–o] ×400, all performed with hematoxylin counterstain, except double-stain sections, which were counterstained with periodic acid–Schiff alone, without hematoxylin, to allow clear visualization of blue immunohistochemical stain). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.