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

Abstract

The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotising inflammation of small blood vessels and crescentic glomerulonephritis. ANCA are thought to play a direct pathogenic role. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation in vitro. However, the role of SYK in vivo is unknown. Here, we studied its role in the pathogenesis of experimental autoimmune vasculitis, a pre-clinical model of myeloperoxidase-ANCA-induced pauci-immune systemic vasculitis in the Wistar Kyoto rat. Up-regulation of SYK expression in inflamed renal and pulmonary tissue during early autoimmune vasculitis was confirmed by immunohistochemical and transcript analysis. R406, the active metabolite of fostamatinib, a small molecule kinase inhibitor with high selectivity for SYK, inhibited ANCA-induced pro-inflammatory responses in rat leucocytes in vitro. In an in vivo study, treatment with fostamatinib for 14 days after disease onset resulted in rapid resolution of urinary abnormalities, significantly improved renal and pulmonary pathology, and preserved renal function. Short-term exposure to fostamatinib did not significantly affect circulating myeloperoxidase-ANCA levels, suggesting inhibition of ANCA-induced inflammatory mechanisms in vivo. Finally, SYK expression was demonstrated within inflammatory glomerular lesions in ANCA-associated glomerulonephritis in patients, particularly within CD68+ve monocytes/macrophages. Thus, our data indicate that SYK inhibition warrants clinical investigation in the treatment of AAV. The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotising inflammation of small blood vessels and crescentic glomerulonephritis. ANCA are thought to play a direct pathogenic role. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation in vitro. However, the role of SYK in vivo is unknown. Here, we studied its role in the pathogenesis of experimental autoimmune vasculitis, a pre-clinical model of myeloperoxidase-ANCA-induced pauci-immune systemic vasculitis in the Wistar Kyoto rat. Up-regulation of SYK expression in inflamed renal and pulmonary tissue during early autoimmune vasculitis was confirmed by immunohistochemical and transcript analysis. R406, the active metabolite of fostamatinib, a small molecule kinase inhibitor with high selectivity for SYK, inhibited ANCA-induced pro-inflammatory responses in rat leucocytes in vitro. In an in vivo study, treatment with fostamatinib for 14 days after disease onset resulted in rapid resolution of urinary abnormalities, significantly improved renal and pulmonary pathology, and preserved renal function. Short-term exposure to fostamatinib did not significantly affect circulating myeloperoxidase-ANCA levels, suggesting inhibition of ANCA-induced inflammatory mechanisms in vivo. Finally, SYK expression was demonstrated within inflammatory glomerular lesions in ANCA-associated glomerulonephritis in patients, particularly within CD68+ve monocytes/macrophages. Thus, our data indicate that SYK inhibition warrants clinical investigation in the treatment of AAV. Translational StatementThe anti-neutrophil cytoplasm antibody (ANCA)–associated vasculitides are a group of rare diseases characterized by the inflammation of blood vessels, crescentic glomerulonephritis, and lung hemorrhage. Current treatments are not completely effective, as many patients develop refractory or relapsing disease, or treatment-related toxicities. This article investigates the role of spleen tyrosine kinase (SYK), an immunoreceptor-associated signaling protein, in the pathogenesis of ANCA vasculitis and its potential as a therapeutic target. In a preclinical model, SYK upregulation was identified at sites of renal and pulmonary inflammation, and treatment with an SYK inhibitor was completely effective in treating established lung and kidney disease, without hematological toxicity. SYK expression is also confirmed in glomerular inflammatory lesions in human ANCA-associated vasculitides. Clinical studies of SYK inhibition in IgA nephropathy are ongoing, and these novel data suggest that this approach should also be considered in ANCA vasculitis. The anti-neutrophil cytoplasm antibody (ANCA)–associated vasculitides are a group of rare diseases characterized by the inflammation of blood vessels, crescentic glomerulonephritis, and lung hemorrhage. Current treatments are not completely effective, as many patients develop refractory or relapsing disease, or treatment-related toxicities. This article investigates the role of spleen tyrosine kinase (SYK), an immunoreceptor-associated signaling protein, in the pathogenesis of ANCA vasculitis and its potential as a therapeutic target. In a preclinical model, SYK upregulation was identified at sites of renal and pulmonary inflammation, and treatment with an SYK inhibitor was completely effective in treating established lung and kidney disease, without hematological toxicity. SYK expression is also confirmed in glomerular inflammatory lesions in human ANCA-associated vasculitides. Clinical studies of SYK inhibition in IgA nephropathy are ongoing, and these novel data suggest that this approach should also be considered in ANCA vasculitis. Spleen tyrosine kinase (SYK) is a cytosolic protein tyrosine kinase that is expressed in most leucocyte populations, where it has diverse immune functions. It has a well characterized role in mediating signaling from classical immunoreceptors such as the B-cell receptor and the Fc receptor, and also from some integrins and C-type lectins.1Mocsai A. Ruland J. Tybulewicz V.L. The SYK tyrosine kinase: a crucial player in diverse biological functions.Nat Rev Immunol. 2010; 10: 387-402Crossref PubMed Scopus (745) Google Scholar Targeting SYK has thus emerged as a potential treatment approach for a variety of immune and inflammatory diseases,2Geahlen R.L. Getting Syk: spleen tyrosine kinase as a therapeutic target.Trends Pharmacol Sci. 2014; 35: 414-422Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar and a number of specific SYK inhibitors are in development.3Thorarensen A. Kaila N. New spleen tyrosine kinase inhibitors: patent applications published during 2011-2013.Pharm Pat Anal. 2014; 3: 523-541Crossref PubMed Scopus (6) Google Scholar We have previously shown that fostamatinib, a small molecule kinase inhibitor with selectivity for SYK, is an effective treatment in experimental models of immune-complex glomerulonephritis.4Smith J. McDaid J.P. Bhangal G. et al.A spleen tyrosine kinase inhibitor reduces the severity of established glomerulonephritis.J Am Soc Nephrol. 2010; 21: 231-236Crossref PubMed Scopus (64) Google Scholar,5McAdoo S.P. Reynolds J. Bhangal G. et al.Spleen tyrosine kinase inhibition attenuates autoantibody production and reverses experimental autoimmune GN.J Am Soc Nephrol. 2014; 25: 2291-2302Crossref PubMed Scopus (37) Google Scholar SYK also mediates proinflammatory responses induced by IgA1 derived from patients with IgA nephropathy,6Kim M.J. McDaid J.P. McAdoo S.P. et al.Spleen tyrosine kinase is important in the production of proinflammatory cytokines and cell proliferation in human mesangial cells following stimulation with IgA1 isolated from IgA nephropathy patients.J Immunol. 2012; 189: 3751-3758Crossref PubMed Scopus (51) Google Scholar and full results from a Phase II clinical study assessing SYK inhibition with fostamatinib in proliferative IgA nephropathy are awaited (NCT02112838). The anti-neutrophil cytoplasm antibody (ANCA)–associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotizing inflammation of small blood vessels and pauci-immune crescentic glomerulonephritis, in which ANCA are thought to play a directly pathogenic role.7Jennette J.C. Falk R.J. Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease.Nat Rev Rheumatol. 2014; 10: 463-473Crossref PubMed Scopus (242) Google Scholar It has previously been shown that SYK activation occurs following ANCA-induced neutrophil activation,8Hewins P. Williams J.M. Wakelam M.J. Savage C.O. Activation of Syk in neutrophils by antineutrophil cytoplasm antibodies occurs via Fcgamma receptors and CD18.J Am Soc Nephrol. 2004; 15: 796-808Crossref PubMed Scopus (57) Google Scholar suggesting that SYK inhibition may be a potential therapeutic approach in AAV, though in vivo data are lacking. Here, we have investigated the effect of SYK inhibition in an experimental model of myeloperoxidase (MPO)-ANCA–induced systemic vasculitis (experimental autoimmune vasculitis [EAV]) that was developed in our laboratory.9Little M.A. Smyth C.L. Yadav R. et al.Antineutrophil cytoplasm antibodies directed against myeloperoxidase augment leukocyte-microvascular interactions in vivo.Blood. 2005; 106: 2050-2058Crossref PubMed Scopus (225) Google Scholar,10Little M.A. Smyth L. Salama A.D. et al.Experimental autoimmune vasculitis: an animal model of anti-neutrophil cytoplasmic autoantibody-associated systemic vasculitis.AmJ Pathol. 2009; 174: 1212-1220Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar It is characterized by ANCA-induced enhancement of leucocyte–endothelial cell interactions and the development of both alveolar hemorrhage and necrotizing glomerulonephritis by 4 weeks after disease induction. In contrast to our previous studies in immune-complex glomerulonephritis, this model has a distinct pauci-immune mechanism of tissue injury, similar to that in AAV. We performed immunohistochemical staining for total (T)- and activated (i.e., phosphorylated [P]-) SYK. In healthy rat lung tissue, this analysis demonstrated that T-SYK was expressed in large airway cuboidal epithelial cells and associated lymphoid tissue (Figure 1a), consistent with previously described patterns of SYK expression in hematopoetic and some epithelial cell types.11Ulanova M. Puttagunta L. Marcet-Palacios M. et al.Syk tyrosine kinase participates in beta1-integrin signaling and inflammatory responses in airway epithelial cells.Am J Physiol Lung Cell Mol Physiol. 2005; 288: L497-L507Crossref PubMed Scopus (86) Google Scholar There was minimal T-SYK detection in alveolar squamous epithelium (Figure 1b). In lung tissue taken from animals 6 weeks after induction of EAV (Figure 1c), alveolar lumens were consolidated with erythrocytes, consistent with the development of lung hemorrhage. In addition, large mononuclear cells with cytoplasmic T-SYK expression were seen. Staining of serial sections identified a population of mononuclear cells positive for ED-1 (the rat homologue of CD68), T-SYK, and P-SYK (Figure 1d–f, respectively) in diseased lung, and dual staining confirmed T-SYK expression in ED-1+ve cells (Figure 1g), suggesting an infiltrating population of monocytes/macrophages expressing activated SYK at sites of alveolar hemorrhage. A small number of T-SYK+ve ED-1-ve cells were also observed, suggesting additional cell populations that express SYK in this model, potentially lymphocytes or neutrophils. As previously described, in normal rat kidney tissue, T-SYK was detected in distal tubular epithelial cells but not in normal glomeruli. In kidney tissue taken from animals with established EAV, T-SYK was detected within inflamed glomeruli, particularly within areas of endocapillary proliferation and crescent formation, whereas there was no SYK detection in unaffected glomeruli (Figure 1h). Upregulation of SYK expression was confirmed by the finding of increased SYK mRNA in diseased renal tissue, by both in situ hybridization (Figure 1i and j) and by real-time quantitative polymerase chain reaction (RT-qPCR; Figure 1k). Dual staining showed co-localization of T-SYK and ED-1+ve cells within inflammatory glomerular lesions (Figure 1l). As observed in lung tissue, a small population of T-SYK+ve ED-1–ve cells was seen in some glomeruli. Staining of serial sections suggested that P-SYK localizes to infiltrating ED-1+ve monocytes/macrophages in and around glomeruli (Figure 1m and n). P-SYK staining in kidney sections was both cytoplasmic and nuclear; SYK is known to have a nuclear localization signal in B lymphocytes,12Zhou F. Hu J. Ma H. et al.Nucleocytoplasmic trafficking of the Syk protein tyrosine kinase.Mol Cell Biol. 2006; 26: 3478-3491Crossref PubMed Scopus (56) Google Scholar and we have previously described nuclear staining for P-SYK in human kidney disease.13McAdoo S.P. Bhangal G. Page T. et al.Correlation of disease activity in proliferative glomerulonephritis with glomerular spleen tyrosine kinase expression.Kidney Int. 2015; 88: 52-60Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar In order to confirm SYK phosphorylation in EAV kidney tissue, we performed immunoblotting for P-SYK in kidney cortex, and showed upregulation compared with control kidney tissue (Figure 1o). These immunohistochemical data suggest that SYK is expressed and activated at sites of renal and pulmonary inflammation in EAV. We therefore sought to examine the effect of SYK inhibition using fostamatinib in vivo. EAV was induced in Wistar Kyoto rats (n = 8 per group) by immunization with recombinant human MPO in complete Freunds adjuvant (CFA). onset of vasculitis was confirmed by the development of and 4 weeks after disease animals were with fostamatinib of or by and for disease severity at 6 after a total of 14 days of treatment with or animals were with and 6 weeks (n = lung and renal at treatment an additional group of animals was after induction of EAV (n = weeks after induction of EAV, the of animals of lung and in this to lung hemorrhage in animals by 6 (Figure treatment resulted in a in the severity of pulmonary as by of lung tissue at the of (Figure and lung hemorrhage and for and and by for cells in lung (Figure and detection and for and = In addition, there was a in ED-1 positive (Figure and and ED-1 for and treatment with fostamatinib to complete of lung hemorrhage in of fostamatinib treatment 4 weeks after disease induction was associated with rapid resolution of urinary during the treatment to there were in both (Figure and and for and = and (Figure and and for and = These were associated with a of renal (Figure and for and = of renal tissue at 4 to treatment confirmed of early proliferative glomerulonephritis and that by 6 weeks to necrotizing and crescentic glomerulonephritis in animals (Figure There was a in proliferative glomerular lesions (Figure and and glomerular for and = and ED-1+ve glomerular (Figure and and cells per glomerular for and = In with glomerular cell analysis of expression (Figure confirmed a in proinflammatory and production with fostamatinib without a effect the expression of cytokines There was a in SYK expression following fostamatinib tyrosine kinase (SYK) inhibition renal in experimental autoimmune vasculitis of glomerular at treatment 4 and at 6 weeks after disease with necrotizing glomerulonephritis and crescent in animals and preserved glomerular after fostamatinib treatment of ED-1 homologue of cells infiltrating glomeruli at treatment and at 6 weeks after disease a with fostamatinib with staining for ED-1 with in expression following treatment with fostamatinib, as by real-time quantitative polymerase chain reaction and expressed as compared to normal kidney data are as by or with to group complete Freunds protein not of this the of this article at Figure Short-term fostamatinib treatment did not significantly affect circulating in this study (Figure and 6 weeks after disease and for and = was there a effect following treatment Figure for in renal tissue demonstrated a pauci-immune of renal and confirmed no in glomerular immune control animals and treatment group (Figure and treatment did not significantly compared to animals (Figure However, there was a in blood cell following fostamatinib treatment compared to consistent with previous that have this to a effect (Figure blood cell 6 weeks after disease and for and = However, blood cell were not of control animals with (Figure there was no in circulating after we observed that SYK inhibition express high of the ANCA P. mechanisms of and to human and 2009; PubMed Scopus Google Scholar and it has been shown that rat neutrophils and proinflammatory cytokines following ANCA G. et of antibodies and glomerular neutrophil activation glomerulonephritis in experimental cytoplasmic antibody PubMed Scopus Google Scholar We confirmed that EAV is to mononuclear and cells by (Figure and that SYK is phosphorylated following stimulation of cells with rat (Figure We sought to examine of SYK inhibition using R406, the active metabolite of fostamatinib, responses in vitro. cells high of following stimulation with control no additional with was associated with a in production (Figure and with and R406, = There was a similar in production of induced by following with (Figure We and have demonstrated that SYK expression is and that SYK is at sites of glomerular inflammation in renal S.P. Bhangal G. Page T. et al.Correlation of disease activity in proliferative glomerulonephritis with glomerular spleen tyrosine kinase expression.Kidney Int. 2015; 88: 52-60Abstract Full Text Full Text PDF PubMed Scopus (25) Google J. Ma et renal in glomerulonephritis spleen tyrosine Pathol. PubMed Scopus Google Scholar previous were using a antibody that is no We therefore have our previous of SYK expression in using an antibody directed against human SYK (Figure SYK was detected within inflammatory lesions in areas of glomerular crescent (Figure and (Figure and inflammation but not within normal or glomeruli (Figure and suggesting that it is a of active Staining of serial sections co-localization of SYK to CD68+ve cells in these inflammatory lesions (Figure and staining of SYK and in cells within glomerular (Figure cells were also observed within the glomerular similar to the of staining observed in rat tissue, which may cell such as that may be during the of The are consistent with our previous that SYK inhibition with fostamatinib is effective in both and treating established crescentic glomerulonephritis in disease J. McDaid J.P. Bhangal G. et al.A spleen tyrosine kinase inhibitor reduces the severity of established glomerulonephritis.J Am Soc Nephrol. 2010; 21: 231-236Crossref PubMed Scopus (64) Google Scholar,5McAdoo S.P. Reynolds J. Bhangal G. et al.Spleen tyrosine kinase inhibition attenuates autoantibody production and reverses experimental autoimmune GN.J Am Soc Nephrol. 2014; 25: 2291-2302Crossref PubMed Scopus (37) Google Scholar These data suggest that fostamatinib may ANCA-induced inflammatory and that it may therefore have therapeutic potential in both pauci-immune and glomerular In addition, these data that the treatment effect of fostamatinib be with significantly compared to our previous in experimental autoimmune glomerulonephritis, and without toxicity. It has been shown that ANCA-induced SYK activation in neutrophils is by signaling Fc receptors for and the P. Williams J.M. Wakelam M.J. Savage C.O. Activation of Syk in neutrophils by antineutrophil cytoplasm antibodies occurs via Fcgamma receptors and CD18.J Am Soc Nephrol. 2004; 15: 796-808Crossref PubMed Scopus (57) Google Scholar and it is that of signaling via both these receptors cells is for the therapeutic effect observed in this In both pulmonary and renal disease in this model are characterized by and the of the rat to of experimental glomerulonephritis has been at in to that control in R. et number in to glomerulonephritis in rats and 2006; PubMed Scopus Google J. Bhangal G. J. et is a of activation and is associated with glomerulonephritis PubMed Scopus Google J. J. et activity and glomerular in experimental glomerulonephritis.J Am Soc Nephrol. 2010; 21: PubMed Scopus Google Scholar In these cells are known to express ANCA and proinflammatory following A. et antibodies and of cell Biol. Google M. cytoplasmic antibodies human to in PubMed Scopus Google Scholar which is cytoplasmic antibodies of and Fcgamma PubMed Scopus Google it is that inhibition of in both neutrophils and is for the in disease severity observed with fostamatinib In with a role for SYK activity in monocytes/macrophages in mediating disease, our and previous immunohistochemical suggest that these cell are the glomerular leucocyte expressing SYK in both EAV and human AAV, cell may be We also observed a in in renal tissue following fostamatinib and is known to to the pathogenesis of experimental glomerulonephritis and it is in the glomeruli of patients with et and play an important role in the inflammatory of crescentic but is in crescent and Scopus Google T. H. et of crescentic glomerulonephritis by antibodies to and J. 10: PubMed Scopus Google M. L. J. expression of in experimental and human Google Scholar urinary of this with disease activity in A. et is a of active renal 2004; PubMed Scopus Google Scholar and urinary has been as a novel in L. et and in the of renal in anti-neutrophil cytoplasmic 35: Google Scholar potential clinical of SYK inhibition in AAV. It is that we did not a effect circulating following fostamatinib treatment in this study, in contrast to our previous in experimental autoimmune glomerulonephritis, a model of disease, in which we observed inhibition of antibody S.P. Reynolds J. Bhangal G. et al.Spleen tyrosine kinase inhibition attenuates autoantibody production and reverses experimental autoimmune GN.J Am Soc Nephrol. 2014; 25: 2291-2302Crossref PubMed Scopus (37) Google Scholar It is that this in the at which treatment was in the experimental autoimmune glomerulonephritis, treatment was after antibody were whereas in this study, treatment was after antibody responses were and did not the of It is known that B-cell and antibody production are SYK via both B-cell and B-cell L. J. et receptor by the B cell receptor signaling Full Text Full Text PDF PubMed Scopus Google J. et al.Syk tyrosine kinase is for B cell antibody responses and B cell 2015; PubMed Scopus Google Scholar is an established in the treatment of for the treatment of autoimmune renal Rev Nephrol. PubMed Scopus Google Scholar and of these B-cell with fostamatinib treatment may additional therapeutic in should also be activity may to some of the therapeutic observed in this In with many small molecule kinase R406, the active metabolite of fostamatinib, has been shown to with a of within the human in J.P. et analysis of kinase inhibitor PubMed Scopus Google M. et of the clinical of 2015; PubMed Scopus Google Scholar in of demonstrated high selectivity for SYK as by phosphorylation of similar isolated kinase H. et an spleen tyrosine kinase inhibitor receptor signaling and reduces immune Pharmacol 2006; PubMed Scopus Google Scholar A of activity against and is and may have to the The effect of fostamatinib which not express SYK, but in which signaling to for A specific of SYK to and glomerular inflammation, is suggested by studies in and with J. Ma et renal in glomerulonephritis spleen tyrosine Pathol. PubMed Scopus Google J. Ma J. et al.Spleen tyrosine kinase glomerular via activation of and in rat PubMed Scopus Google Ma J. inhibitor of spleen tyrosine kinase experimental crescentic J PubMed Scopus Google Scholar In this study, we have confirmed our previous and of that SYK expression is and that SYK is at sites of glomerular inflammation in renal S.P. Bhangal G. Page T. et al.Correlation of disease activity in proliferative glomerulonephritis with glomerular spleen tyrosine kinase expression.Kidney Int. 2015; 88: 52-60Abstract Full Text Full Text PDF PubMed Scopus (25) Google J. Ma et renal in glomerulonephritis spleen tyrosine Pathol. PubMed Scopus Google Scholar additional in vivo data that SYK to disease and that clinical studies of SYK inhibition should be the rapid onset of therapeutic effect observed in the experimental SYK inhibition may be a particularly for rapid control of inflammation in AAV, potentially as a via its cell functions. has shown biological activity and an in clinical studies in A. R. et of with a Syk kinase a PubMed Scopus Google A. et spleen tyrosine kinase inhibitor for J 2010; PubMed Scopus Google Scholar and immune J. et for the treatment of and immune of J PubMed Scopus Google Scholar such that the was by the and for treatment of the The results of a II clinical study of SYK inhibition in IgA nephropathy were in at the of in This study did not a in a of in in the suggested in patients the study with of This that SYK inhibition may be an effective treatment in inflammatory of glomerulonephritis, such as ANCA-associated glomerulonephritis, and our in this preclinical model suggest that this approach should be in clinical studies of AAV. A of in the animal were by the and were in with the and the of In tissue were by the number in with and its active R406, were by and The of these have been H. et an spleen tyrosine kinase inhibitor receptor signaling and reduces immune Pharmacol 2006; PubMed Scopus Google A. et spleen tyrosine kinase inhibitor disease and in PubMed Scopus Google Scholar in vivo animals were with fostamatinib or by and autoimmune vasculitis was induced by Wistar Kyoto rats (n = with human MPO in with was days and as previously M.A. Smyth L. Salama A.D. et al.Experimental autoimmune vasculitis: an animal model of anti-neutrophil cytoplasmic autoantibody-associated systemic vasculitis.AmJ Pathol. 2009; 174: 1212-1220Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar were 6 weeks after disease induction. 4 experimental animals were for severity of urinary at this to or fostamatinib animals were with and 6 weeks (n = at treatment an additional group of animals were after induction of EAV (n = was performed using the antibodies rat T-SYK and human T-SYK rat P-SYK rat ED-1 and human In situ hybridization for SYK mRNA was performed using Cell Lung hemorrhage was by at the of and by of and ED-1+ve cells by using analysis. was by and by the A. Bhangal G. McDaid J. et of protein kinase is effective in the treatment of experimental crescentic glomerulonephritis and but not or Am Soc Nephrol. PubMed Scopus (64) Google Scholar glomerular injury, glomeruli in kidney sections were as normal or and results were expressed as the for were as and the number of positive cells per glomerular were in glomeruli, with results expressed as the for of was performed in a was in by to leucocytes was confirmed by antibodies were detected kidney sections using a and blood cell were in blood using an from renal was to using was performed using are in the was in and were using the to of renal or cells were by to and with the following T-SYK and P-SYK rat cells were with 4 rat for with or control isolated from EAV or healthy inhibition cells were with for after and to production was using a and production was by were to of the biological analysis was using data are as per group was by and with is an of and for has a from and has a with has from and and is the of an clinical of an SYK inhibitor in IgA nephropathy by has with and the no This was by Clinical a Clinical and a is by the from and the and of We from the for of the were in at the of in 2014; the of in published in The and the and ANCA in with

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