Journal: Kidney International

Article Title: Spleen tyrosine kinase inhibition is an effective treatment for established vasculitis in a pre-clinical model

doi: 10.1016/j.kint.2019.12.014

Figure Lengend Snippet: 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 .

Article Snippet: 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).

Techniques: Immunohistochemical staining, Staining, Expressing, Double Staining, In Situ Hybridization, Adjuvant, Comparison, MANN-WHITNEY, Activation Assay, Western Blot

Journal: Kidney International

Article Title: Spleen tyrosine kinase inhibition is an effective treatment for established vasculitis in a pre-clinical model

doi: 10.1016/j.kint.2019.12.014

Figure Lengend Snippet: Spleen tyrosine kinase (SYK) inhibition reduces the severity of lung hemorrhage in experimental autoimmune vasculitis (EAV). ( a ) Lung hemorrhage severity scores in control, vehicle, and fostamatinib (Fosta)-treated rats, assessed at treatment initiation (week 4 [W4]) and at cull 6 weeks after disease induction, with lower panel ( b ) showing representative macroscopic lung pathology. ( c ) Quantification for hemosiderin deposition in lung tissue at treatment initiation (W4) and 6 weeks after disease induction, with representative photomicrographs ( d ) demonstrating staining for Perls’ Prussian Blue (without counterstain; original magnification ×200). ( e ) Quantification for ED-1 (rat homologue of CD68)–positive cells in lung tissue at treatment initiation (W4) and 6 weeks after disease induction, with ( f ) representative photomicrographs of ED-1 staining in lung tissue (with hematoxylin counterstain; original magnification ×200). All data are reported as median ± interquartile range, statistical comparison by Kruskal-Wallis test, with Dunn’s post-test comparison to vehicle group (lower indicator); ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. CFA, complete Freunds adjuvant; LH, lung hemorrhage; ns, not significant. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org .

Article Snippet: 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).

Techniques: Inhibition, Staining, Comparison, Adjuvant

Journal: Kidney International

Article Title: Spleen tyrosine kinase inhibition is an effective treatment for established vasculitis in a pre-clinical model

doi: 10.1016/j.kint.2019.12.014

Figure Lengend Snippet: Spleen tyrosine kinase (SYK) inhibition improves renal histopathology in experimental autoimmune vasculitis (EAV). ( a ) Quantification of glomerular abnormalities at treatment initiation (week 4 [W4]) and at 6 weeks after disease induction, with ( c ) representative photomicrographs demonstrating focal necrotizing glomerulonephritis and crescent formation in vehicle-treated animals and preserved glomerular histology after fostamatinib (Fosta) treatment (periodic acid–Schiff [PAS]–stained sections, original magnification ×400). ( b ) Quantification of ED-1 (rat homologue of CD68)–positive cells infiltrating glomeruli at treatment initiation (W4) and at 6 weeks after disease induction, showing a dose-dependent reduction with fostamatinib treatment, with representative photomicrographs demonstrating immunoperoxidase staining for ED-1 ( b ; with hematoxylin counterstain; original magnification ×400). ( d ) Heat-map indicating changes in intra-renal gene expression following treatment with fostamatinib, as determined by real-time quantitative polymerase chain reaction and expressed as fold-change compared to normal kidney tissue. All data are reported as median ± interquartile range; statistical comparison by Mann-Whitney or Kruskal-Wallis test, with Dunn’s post-test comparison to vehicle group (lower indicator); ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.0001. CCL3, C–C motif chemokine ligand 3; CFA, complete Freunds adjuvant; MCP-1, monocyte chemoattractant protein 1; MMP9, matrix metallopeptidase 9; ns, not significant; TNF, tumor necrosis factor. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org .

Article Snippet: 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).

Techniques: Inhibition, Histopathology, Staining, Immunoperoxidase Staining, Expressing, Real-time Polymerase Chain Reaction, Comparison, MANN-WHITNEY, Adjuvant

Journal: Kidney International

Article Title: Spleen tyrosine kinase inhibition is an effective treatment for established vasculitis in a pre-clinical model

doi: 10.1016/j.kint.2019.12.014

Figure Lengend Snippet: Short-term fostamatinib (Fosta) treatment has minimal effects on myeloperoxidase–anti-neutrophil cytoplasm antibody (MPO-ANCA) production in experimental autoimmune vasculitis (EAV), although it inhibits MPO-ANCA–induced cellular responses in vitro . ( a ) MPO-ANCA titres from day of disease induction (week 0 [W0]) to week 6 (W6), with the fostamatinib treatment period shaded in gray, showing no significant effect of treatment on circulating autoantibody levels. ( b ) Individual MPO-ANCA levels at W4 and W6. ( c ) Quantification of immunofluorescence (IF) staining for deposited IgG in glomeruli at treatment initiation (W4) and at 6 weeks after disease induction, confirming no difference in deposited IgG before or after fostamatinib treatment, with ( d ) representative immunofluorescence staining. Positive control tissue from animals after induction of nephrotoxic nephritis (NTN) included for comparison (all images, original magnification ×400). ( e,f ) Hematological indices following fostamatinib treatment in EAV, including ( e ) hemoglobin concentration and ( f ) white blood cell (WBC) count in each treatment group 6 weeks after disease induction. Fostamatinib treatment did not significantly alter hemoglobin concentrations. WBC counts were modestly reduced in fostamatinib-treated animals compared to vehicle-treated controls, although not beyond those of complete Freunds adjuvant (CFA)–alone immunized animals. ( g ) Indirect immunofluorescence on ethanol-fixed undifferentiated rat bone marrow cells using EAV and CFA control serum, confirming reactivity of EAV serum for both mononuclear and polymorphonuclear cells. ( h ) Immunoblotting confirms upregulation of SYK phosphorylation in undifferentiated bone marrow cells following priming with tumor necrosis factor α (TNFα) and stimulation with MPO-ANCA IgG. ( i,j ) Cellular responses by undifferentiated rat bone marrow cells following stimulation with TNFα, MPO-ANCA IgG, and control IgG (cIgG), alone or in combination, and following pretreatment with the active metabolite of fostamatinib, R406, or vehicle, showing ( i ) dose-dependent reduction in monocyte chemoattractant protein–1 (MCP-1) production (standardized results expressed as mean of 4 replicate experiments), and ( j ) inhibition of reactive oxygen species (ROS) generation, following R406 treatment. R406 exposure did not affect cell survival ( Supplementary Figure S2 ). All data are reported as median ± interquartile range, statistical comparison by Kruskal-Wallis test, with Dunn’s post-test comparison to vehicle group (lower indicator); ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant; SYK, spleen tyrosine kinase. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org .

Article Snippet: 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).

Techniques: In Vitro, Immunofluorescence, Staining, Positive Control, Comparison, Concentration Assay, Adjuvant, Western Blot, Inhibition

Journal: Kidney International

Article Title: Spleen tyrosine kinase inhibition is an effective treatment for established vasculitis in a pre-clinical model

doi: 10.1016/j.kint.2019.12.014

Figure Lengend Snippet: Spleen tyrosine kinase (SYK) is expressed in inflammatory lesions in human anti-neutrophil cytoplasm antibody (ANCA)–associated glomerulonephritis. Immunohistochemical staining for total (T)-SYK in ANCA-associated glomerulonephritis (AAGN) demonstrates SYK expression within inflammatory glomerular lesions including ( a ) crescents, but not within ( b ) normal or ( c ) sclerotic glomeruli. Staining of paired sections ( d–i ) for CD68 and T-SYK indicates significant co-localization of T-SYK to CD68-positive macrophages ( d,e ) within areas of glomerular fibrinoid necrosis, ( f,g ) extra-capillary proliferation, and ( h,i ) periglomerular inflammation. ( j ) Double staining for T-SYK (brown) and CD68 (blue) indicates distal tubular epithelial cell staining for T-SYK, as previously described (long solid arrowhead). Within glomeruli, there was significant co-localization of T-SYK and CD68 (short solid arrowhead) in cells within crescents. In addition, occasional T-SYK+ CD68– cells were observed (short open arrowhead). Photomicrographs, original magnification ×200 ( a–i ) and ×400 ( j ), all performed with hematoxylin and periodic acid–Schiff counterstain, except ( j ), which was counterstained with periodic acid–Schiff alone, without hematoxylin, to allow clear visualization of the blue immunohistochemical stain. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org .

Article Snippet: 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).

Techniques: Immunohistochemical staining, Staining, Expressing, Double Staining