ABSTRACT
Introduction: We reported increased spleen tyrosine kinase (SYK) expression in kidney biopsies of patients with IgA nephropathy (IgAN) and that inhibition of SYK reduces inflammatory cytokines production from IgA stimulated mesangial cells. Methods: This study was a double-blind, randomized, placebo-controlled phase 2 trial of fostamatinib (an oral SYK inhibitor) in 76 patients with IgAN. Patients were randomized to receive placebo, fostamatinib at 100 mg or 150 mg twice daily for 24 weeks on top of maximum tolerated dose of renin-angiotensin system inhibitors. The primary end point was reduction of proteinuria. Secondary end points included change from baseline in estimated glomerular filtration rate (eGFR) and kidney histology. Results: Although we could not detect significant reduction in proteinuria with fostamatinib overall, in a predetermined subgroup analysis, there was a trend for dose-dependent reduction in median proteinuria (from baseline to 24 weeks by 14%, 27%, and 36% in the placebo, fostamatinib 100 mg, and 150 mg groups, respectively) in patients with baseline urinary protein-to-creatinine ratios (UPCR) more than 1000 mg/g. Kidney function (eGFR) remained stable in all groups. Fostamatinib was well-tolerated. Side effects included diarrhea, hypertension, and increased liver enzymes. Thirty-nine patients underwent repeat biopsy showing reductions in SYK staining associated with therapy at low dose (-1.5 vs. 1.7 SYK+ cells/glomerulus in the placebo group, P < 0.05). Conclusions: There was a trend toward reduction in proteinuria with fostamatinib in a predefined analysis of high risk patients with IgAN despite maximal care, as defined by baseline UPCR greater than 1000 mg/g. Further study may be warranted.
ABSTRACT
Experimental autoimmune vasculitis (EAV) is a model of antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) induced by immunisation of susceptible rat strains with myeloperoxidase (MPO). Animals develop circulating MPO-ANCA, pulmonary haemorrhage, and glomerulonephritis, although renal injury is mild and recovers spontaneously without treatment. In this study we aimed to augment the severity of glomerulonephritis. Following induction of EAV on day 0, a sub-nephritogenic dose of nephrotoxic serum (NTS) containing heterologous antibodies to glomerular basement membrane was administered on day 14. This resulted in a significant increase in disease severity at day 28 compared to MPO immunisation alone - with more urinary abnormalities, infiltrating glomerular leucocytes, and crescent formation that progressed to glomerular and tubulointerstitial scarring by day 56, recapitulating important features of human disease. Importantly, the glomerulonephritis remained pauci-immune, and was strictly dependent on the presence of autoimmunity to MPO, as there was no evidence of renal disease following administration of sub-nephritogenic NTS alone or after immunisation with a control protein in place of MPO. Detailed phenotyping of glomerular leucocytes identified an early infiltrate of non-classical monocytes following NTS administration that, in the presence of autoimmunity to MPO, may initiate the subsequent influx of classical monocytes which augment glomerular injury. We also showed that this model can be used to test novel therapeutics by using a small molecule kinase inhibitor (fostamatinib) that rapidly attenuated both glomerular and pulmonary injury over a 4-day treatment period. We believe that this enhanced model of MPO-AAV will prove useful for the study of glomerular leucocyte behaviour and novel therapeutics in AAV in the future. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Autoantigens/immunology , Disease Models, Animal , Glomerulonephritis/immunology , Peroxidase/immunology , Animals , Autoantibodies/immunology , Glomerular Basement Membrane/immunology , Male , RatsABSTRACT
BACKGROUND: To investigate whether serum chitinase-3-like 1 protein (YKL-40) is associated with disease activity in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). METHODS: ELISA was performed in serum samples from AAV patients who were enrolled in our prospective observational cohort to estimate levels of YKL-40. Birmingham vasculitis activity score (BVAS) (version 3), five factor score (FFS), and short form-36 (SF-36), as well as clinical and laboratory data were collected. Kidney expression of YKL-40 was assessed by immunohistochemical staining using renal biopsy tissues from ANCA-associated glomerulonephritis patients (AAGN). Severe AAV and FFS were defined as BVAS ≥ 12 and FFS ≥ 2, and the correlations between laboratory variables, BVAS, FFS, and SF-36 score were assessed using linear regression analysis. The optimal cut-off of serum YKL-40 for severe AAV and high FFS was calculated using the receiver operator characteristic curve analysis. RESULTS: Of the included 60 patients, 32 (53.3%), 17 (28.3%), and 11 (18.3%) were classified as microscopic polyangiitis, granulomatosis with polyangiitis, and eosinophilic granulomatosis with polyangiitis. The median BVAS and FFS were 7.0 and 1.0, whereas the mean SF-36 physical and mental component scores were 50.5 and 58.3. Serum YKL-40 level was higher in patients with severe AAV and high FFS compared to those without (p = 0.007 and p < 0.001); multivariable linear regression analysis revealed that serum YKL-40 was independently associated with BVAS, FFS, and SF-36 scores. On kidney tissues obtained from AAGN patients, strong cytoplasmic staining of YKL-40 was found in cells present in inflammatory lesions. In addition, AAV patients had higher levels of serum YKL-40 compared to those with systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, and healthy control. The proportion of patients having severe AAV and high FFS was significantly higher in those with serum YKL-40 > 221.3 ng/mL and > 227.1 ng/mL than those without (relative risk 2.852 and 7.000). In 12 patients with serial YKL-40 testing, 11 patients (91.7%) exhibited a reduction in serum YKL-40 levels following a decrease in disease activity (p < 0.001). CONCLUSION: Our findings suggest that serum YKL-40 may be a clinically useful biomarker to assess AAV disease activity. TRIAL REGISTRATION: Retrospectively registered.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis , Chitinases , Churg-Strauss Syndrome , Granulomatosis with Polyangiitis , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Antibodies, Antineutrophil Cytoplasmic , Biomarkers , Humans , Prospective StudiesABSTRACT
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.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis , Glomerulonephritis , Animals , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/drug therapy , Antibodies, Antineutrophil Cytoplasmic , Glomerulonephritis/drug therapy , Humans , Peroxidase , Rats , Syk KinaseABSTRACT
BACKGROUND: Diabetic nephropathy is the leading cause of end stage kidney disease worldwide. The pathogenesis of this disease remains elusive and multiple factors have been implicated. These include the effects of hyperglycaemia, haemodynamic and metabolic factors, and an inflammatory process that stimulates cellular signalling pathways leading to disease progression and severe fibrosis. Fibronectin (Fn) is an important protein of the extracellular matrix that is essential in fibrosis and its presence in increased amounts has been identified in the kidney in diabetic nephropathy. METHODS: Proximal tubuloepithelial (HK-2) cells were stimulated with high glucose (30 mM D-glucose) or glycated albumin (500 µg/mmol) + 4 mM D-glucose or their controls, Mannitol (26 mM + 4 mM D-glucose) and 4 mM D-glucose, respectively. Following 48 h of stimulation the supernatant was collected and MTT [3-(4,5-dimethylthiazole-2,5-diphenyltetrazolium bromide] assay performed to assess cell viability. HK-2 cells were also stimulated in the above environments with recombinant CCL18 (rCCL18) or MCP-1 (rMCP-1) for 48 h with quantification of Fn levels using ELISA. RESULTS: Co-stimulation of HK-2 cells with high concentrations of glucose and rCCL18 significantly increased Fn (p < 0.001), in comparison to high concentrations of glucose alone. HK-2 cells stimulated with glycated albumin consistently produced Fn and this did not alter following co-stimulation with rCCL18 or rMCP-1. CONCLUSION: This study demonstrates how stimulation with a specific chemokine CCL18 in high glucose upregulates the production of Fn from proximal tubuloepithelial cells. This may be relevant to the development of renal fibrosis in diabetic nephropathy.
ABSTRACT
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.
Subject(s)
Inflammation/immunology , Inflammation/prevention & control , Macrophage Migration-Inhibitory Factors/immunology , Molecular Targeted Therapy/methods , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Blotting, Western , Dexamethasone/immunology , Dexamethasone/therapeutic use , Disease Models, Animal , Enterocolitis/immunology , Enterocolitis/metabolism , Enterocolitis/prevention & control , Flow Cytometry , Glomerulonephritis/immunology , Glomerulonephritis/metabolism , Glomerulonephritis/prevention & control , Glucocorticoids/immunology , Glucocorticoids/therapeutic use , Humans , Inflammation/metabolism , Macrophage Migration-Inhibitory Factors/chemistry , Macrophage Migration-Inhibitory Factors/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Oxidation-Reduction , Protein Conformation , Protein Isoforms/chemistry , Protein Isoforms/immunology , Protein Isoforms/metabolism , Rabbits , Rats, Inbred WKYABSTRACT
Spleen tyrosine kinase (SYK) is an important component of the intracellular signaling pathway for various immunoreceptors. Inhibition of SYK has shown promise in preclinical models of autoimmune and glomerular disease. However, the description of SYK expression in human renal tissue, which would be desirable ahead of clinical studies, is lacking. Here we conducted immunohistochemical analysis for total and phosphorylated SYK in biopsy specimens from >120 patients with a spectrum of renal pathologies, including thin basement membrane lesion, minimal change disease, membranous nephropathy, IgA nephropathy, lupus nephritis, ANCA-associated glomerulonephritis, antiglomerular basement membrane disease, and acute tubular necrosis. We found significant SYK expression in proliferative glomerulonephritis and that glomerular expression levels correlated with presenting serum creatinine and histological features of disease activity that predict outcome in IgA nephropathy, lupus nephritis, ANCA-associated glomerulonephritis, and antiglomerular basement membrane disease. SYK was phosphorylated within pathological lesions, such as areas of extracapillary and endocapillary proliferation, and appeared to localize to both infiltrating leucocytes and to resident renal cells within diseased glomeruli. Thus SYK is associated with the pathogenesis of proliferative glomerulonephritides, suggesting that these conditions may respond to SYK inhibitor treatment.
Subject(s)
Capillaries/chemistry , Glomerulonephritis/enzymology , Intracellular Signaling Peptides and Proteins/analysis , Kidney Glomerulus/enzymology , Leukocytes/chemistry , Protein-Tyrosine Kinases/analysis , Antigens, CD/analysis , Antigens, Differentiation, Myelomonocytic/analysis , Creatinine/blood , Epithelial Cells/chemistry , Glomerulonephritis/blood , Glomerulonephritis/pathology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Kidney Tubules, Distal/chemistry , Neovascularization, Pathologic/blood , Neovascularization, Pathologic/enzymology , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Syk Kinase , Up-RegulationABSTRACT
Spleen tyrosine kinase (SYK) has an important role in immunoreceptor signaling, and SYK inhibition has accordingly attenuated immune-mediated injury in several in vivo models. However, the effect of SYK inhibition on autoantibody production remains unclear, and SYK inhibition has not been studied in an autoimmune model of renal disease. We, therefore, studied the effect of SYK inhibition in experimental autoimmune GN, a rodent model of antiglomerular basement membrane disease. We show glomerular SYK expression and activation by immunohistochemistry in both experimental and clinical disease, and we show that treatment with fostamatinib, a small molecule kinase inhibitor selective for SYK, completely prevents the induction of experimental autoimmune GN. In established experimental disease, introduction of fostamatinib treatment led to cessation of autoantibody production, reversal of renal injury, preservation of biochemical renal function, and complete protection from lung hemorrhage. B cell ELISpot and flow cytometric analysis suggest that short-term fostamatinib treatment inhibits the generation and activity of antigen-specific B cells without affecting overall B-cell survival. Additionally, fostamatinib inhibited proinflammatory cytokine production by nephritic glomeruli ex vivo and cultured bone marrow-derived macrophages in vitro, suggesting additional therapeutic effects independent of effects on autoantibody production that are likely related to inhibited Fc receptor signaling within macrophages in diseased glomeruli. Given these encouraging results in an in vivo model that is highly applicable to human disease, we believe clinical studies targeting SYK in GN are now warranted.
Subject(s)
Anti-Glomerular Basement Membrane Disease/immunology , Anti-Glomerular Basement Membrane Disease/prevention & control , Antibody Formation/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Oxazines/therapeutic use , Protein-Tyrosine Kinases/metabolism , Pyridines/therapeutic use , Aminopyridines , Animals , Autoantibodies/blood , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Morpholines , Oxazines/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Pyrimidines , Rats, Inbred WKY , Spleen/drug effects , Syk KinaseABSTRACT
BACKGROUND: Encapsulating peritoneal sclerosis (EPS) is a rare but devastating complication of long-term peritoneal dialysis (PD). There is no well-validated method for predicting which patients will develop the condition, although known risk factors include long duration of PD, high glucose exposure and lack of residual renal function. We have investigated whether dialysate cytokines (MCP-1 (monocyte chemotactic protein-1), CCL18 (pulmonary and activation-regulated cytokine, PARC), IL-6 (interleukin-6), CCL15 (leukotactin) and angiogenin) could be used to predict the onset of EPS more effectively than known clinical risk factors. METHODS: Samples of dialysate and clinical data were prospectively collected from 151 patients at the West London Renal center between 2003 and 2010. Dialysate cytokine levels were measured using the enzyme-linked immunoabsorbant assay (ELISA) technique. Encapsulating peritoneal sclerosis subsequently developed in 17 patients during a follow-up period of 27 - 113 months. Cytokines found at higher levels in dialysate of pre-EPS patients were investigated as candidate predictors of EPS using logistic regression analysis. RESULTS: Dialysate IL-6, MCP-1 and CCL15 were significantly higher in patients who subsequently developed EPS; however, a logistic regression model using dialysate cytokines to predict EPS was no better than a model using well-recognized clinical markers (length of time on PD and membrane transport status). CONCLUSIONS: Although MCP-1, IL-6 and CCL15 were found at higher levels in the dialysate of patients who subsequently developed EPS, dialysate levels of these cytokines do not improve prediction of future EPS above a model using known clinical risk factors.
Subject(s)
Cytokines/metabolism , Dialysis Solutions/analysis , Peritoneal Dialysis/adverse effects , Peritoneal Fibrosis/diagnosis , Aged , Biomarkers/analysis , Chemokine CCL2/analysis , Chemokines, CC/analysis , Cohort Studies , Cytokines/analysis , Enzyme-Linked Immunosorbent Assay , Female , Humans , Interleukin-6/analysis , Logistic Models , Macrophage Inflammatory Proteins/analysis , Male , Middle Aged , Peritoneal Dialysis/methods , Peritoneal Fibrosis/etiology , Predictive Value of Tests , Prognosis , Prospective Studies , ROC Curve , Risk Assessment , Severity of Illness IndexABSTRACT
BACKGROUND: The ATP-sensitive P2X7 receptor (P2X7R) has been shown to contribute to renal injury in nephrotoxic nephritis, a rodent model of acute glomerulonephritis, and in unilateral ureteric obstruction (UUO), a rodent model of chronic interstitial inflammation and fibrosis. Renal tubular cells, endothelial cells and macrophages also express the closely related P2X4 receptor (P2X4R), which is chromosomally co-located with P2X7R and has 40% homology; it is also pro-inflammatory and has been shown to interact with P2X7R to modulate its pro-apoptotic and pro-inflammatory effects. Therefore, we chose to explore the function of P2X4R in the UUO model of renal injury using knockout mice. We hypothesized that UUO-induced tubulointerstitial damage and fibrosis would also be attenuated in P2X4R(-/-) mice. METHOD: P2X4R(-/-) and wild-type (WT) mice were subjected to either UUO or sham operation. Kidney samples taken on Days 7 and 14 were evaluated for renal inflammation and fibrosis, and expression of pro-fibrotic factors. RESULTS: To our surprise, the obstructed kidney in P2X4R(-/-) mice showed more severe renal injury, more collagen deposition (picrosirius red staining, increase of 53%; P < 0.05) and more type I collagen staining (increase of 107%; P < 0.01), as well as increased mRNA for TGF-ß (increase of 102%, P < 0.0005) and CTGF (increase of 157%; P < 0.05) by Day 14, compared with the UUO WT mice. CONCLUSION: These findings showed that lack of P2X4R expression leads to increased renal fibrosis, and increased expression of TGF-ß and CTGF in the UUO model.
Subject(s)
Kidney/pathology , Nephritis, Interstitial/physiopathology , Receptors, Purinergic P2X4/physiology , Ureteral Obstruction/physiopathology , Animals , Blotting, Western , Cells, Cultured , Collagen Type I/metabolism , Connective Tissue Growth Factor/genetics , Disease Models, Animal , Fibrosis/pathology , Immunoenzyme Techniques , Kidney/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nephritis, Interstitial/genetics , Nephritis, Interstitial/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Transforming Growth Factor beta/genetics , Ureteral Obstruction/genetics , Ureteral Obstruction/metabolismABSTRACT
P2RX7, a mediator of IL-1ß and IL-18 processing and release, is a ligand-gated cation channel that is expressed by macrophages. In experimental Crgn, P2RX7 deficiency attenuates renal injury, but the underlying mechanism is unknown. Here, we show that P2RX7 levels and the expression of several genes belonging to the Nlrp3-inflammasome pathway are up-regulated in the macrophages of the WKY rat, a strain uniquely susceptible to macrophage-dependent NTN. Importantly, following P2RX7 activation, WKY BMDMs produce markedly increased levels of active caspase-1, IL-1ß, and IL-18 when compared with the NTN-resistant LEW rat BMDMs. P2RX7 and active IL-1ß, IL-18, and caspase-1 protein levels were markedly increased in the WKY nephritic glomeruli 4 days following induction of NTN, and the use of a P2RX7 antagonist reduced the levels of secreted active IL-1ß. Interestingly, the post-translational control of P2RX7-mediated inflammasome activation is under the genetic regulation of two previously identified Crgn quantitative trait loci in the BMDMs and nephritic glomeruli of the WKY rat. In conclusion, we propose a novel mechanism, whereby genetically determined P2RX7 levels in macrophages regulate Nlrp3-inflammasome activation and susceptibility to Crgn.
Subject(s)
Genetic Predisposition to Disease , Glomerulonephritis/metabolism , Inflammasomes/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Purinergic P2X7/metabolism , Animals , Blotting, Western , Carrier Proteins , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Gene Knockout Techniques , Glomerulonephritis/genetics , Inflammasomes/genetics , Macrophages/metabolism , Male , NLR Family, Pyrin Domain-Containing 3 Protein , Rats , Rats, Inbred WKY , Real-Time Polymerase Chain Reaction , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Purinergic P2X7/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In experimental autoimmune glomerulonephritis (EAG), a model of Goodpasture's disease, Wistar Kyoto (WKY) rats immunized with collagenase-solubilized glomerular basement membrane (GBM) or the recombinant NC1 domain of the α3 chain of type IV collagen [α3(IV)NC1] develop anti-GBM antibodies and focal necrotizing glomerulonephritis with crescent formation. However, Lewis (LEW) rats, which share the same major histocompatibility complex (MHC) haplotype, are resistant to EAG development. A genome-wide linkage analysis of backcrossed animals with EAG revealed a major quantitative trait locus (QTL) on rat chromosome 13 (LOD = 3.9) linked to the percentage of glomerular crescents. To investigate the role of this QTL in EAG induction, reciprocal congenic rats were generated (LEW.WCrgn1 congenic and WKY.LCrgn1 congenic), immunized with recombinant rat α3(IV)NC1, and assessed for EAG development. WKY.LCrgn1 rats showed a marked reduction in albuminuria, severity of crescentic nephritis, and number of glomerular macrophages compared with WKY controls. No reduction in antibody levels was observed. However, LEW.WCrgn1 rats were resistant to EAG development, as were LEW controls. Macrophage activation in vitro was assessed in parental and congenic rat bone marrow-derived macrophages (BMDMs). WKY.LCrgn1 BMDMs showed a significant reduction in Fc receptor-mediated oxidative burst, phagocytosis of opsonised polystyrene beads, and LPS-induced levels of MCP-1 secretion and iNOS mRNA expression compared with WKY rats. These results confirm the importance of Crgn1 on chromosome 13 in EAG susceptibility, mediated partly through differences in Fc receptor-mediated macrophage activation.
Subject(s)
Anti-Glomerular Basement Membrane Disease/genetics , Animals , Animals, Congenic , Anti-Glomerular Basement Membrane Disease/immunology , Anti-Glomerular Basement Membrane Disease/pathology , Autoantibodies/biosynthesis , Autoantigens/immunology , Chemokine CCL2/biosynthesis , Collagen Type IV/immunology , Female , Genetic Predisposition to Disease , Genome-Wide Association Study , Glomerular Basement Membrane/immunology , Kidney Glomerulus/pathology , Macrophage Activation/genetics , Macrophages/pathology , Male , Nitric Oxide Synthase Type II/metabolism , Phagocytosis/genetics , Phagocytosis/immunology , Phenotype , Quantitative Trait Loci/genetics , Rats , Rats, Inbred Lew , Rats, Inbred WKY , Receptors, Fc/immunology , Recombinant Proteins/immunology , Respiratory Burst/genetics , Respiratory Burst/immunology , Species SpecificityABSTRACT
AIM: The cyclin-dependent kinase inhibitor, seliciclib (R-roscovitine, CYC202), has anti-proliferative activity through its inhibition of cyclin-dependent kinase 2. We hypothesized that treatment with seliciclib would reduce glomerular macrophage numbers and glomerular crescent formation in experimental crescentic glomerulonephritis even when treatment is started after onset of disease. METHOD: Nephrotoxic nephritis (NTN) was induced in Wistar Kyoto rats. In experiment 1, seliciclib (150 mg/kg per day) was given by oral gavage from 1 h before induction of NTN and continued to day 14. In experiment 2, treatment was started on day 4 of NTN and continued to day 14 in order to examine the effect of seliciclib in established glomerulonephritis. RESULTS: In experiment 1, seliciclib reduced proteinuria (119.5 ± 13.9 vs 191.4 ± 18.8 mg/day, P < 0.01), serum creatinine (54.0 ± 3.0 vs 81.0 ± 2.5 µmol/L, P < 0.005) and glomerular crescent score (23.9 ± 2.1 vs 44.6 ± 2.2, P < 0.005) in comparison with controls. In experiment 2, seliciclib ameliorated established glomerulonephritis, with reduction in proteinuria (58 ± 16 vs 165 ± 13 mg/day, P < 0.005), serum creatinine (39 ± 3 vs 62 ± 5 µmol/L, P < 0.05), glomerular macrophage numbers (6.8 ± 2.5 vs 18.5 ± 1.2 ED1+ cells per glomerular cross section, P < 0.05), glomerular cell proliferation (1.2 ± 0.37 vs 4.2 ± 0.80 bromodeoxyuridine (BrdU)+ cells per glomerular section, P < 0.05) and crescent score (10.8 ± 1.6 vs 43.9 ± 1.4, P < 0.05), in comparison with the controls. CONCLUSION: Seliciclib is effective in both prevention and treatment of established crescentic glomerulonephritis in Wistar Kyoto rats, in association with a reduction in the number of glomerular macrophages. We suggest that seliciclib, or other cyclin-dependent kinase inhibitors, may represent a novel therapeutic approach for patients with proliferative glomerulonephritis.
Subject(s)
Cyclin-Dependent Kinases/antagonists & inhibitors , Glomerulonephritis/drug therapy , Kidney Glomerulus/drug effects , Macrophages/drug effects , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Administration, Oral , Animals , Cell Proliferation/drug effects , Creatinine/blood , Cyclin-Dependent Kinases/metabolism , Disease Models, Animal , Glomerulonephritis/enzymology , Glomerulonephritis/pathology , Glomerulonephritis/prevention & control , Kidney Glomerulus/enzymology , Kidney Glomerulus/pathology , Macrophages/pathology , Male , Protein Kinase Inhibitors/administration & dosage , Proteinuria/drug therapy , Proteinuria/enzymology , Purines/administration & dosage , Rats , Rats, Inbred WKY , Roscovitine , Severity of Illness Index , Time FactorsABSTRACT
BACKGROUND: The effect of interleukin-11 (IL-11) on transforming growth factor-ß (TGF-ß) is controversial and has not been examined in renal diseases. In this study, we (i) characterised the up-regulation of TGF-ß1, phospho-p38 MAPK (p-p38 MAPK) and extracellular matrix during pathogenesis of glomerulonephritis and (ii) examined the effect of rhIL-11 on these processes in vivo. METHODS: Following induction of nephrotoxic nephritis, expression of TGF-ß1, alpha-smooth muscle actin (alpha-SMA), fibronectin and p-p38 MAPK was detected in the kidney. Rats were treated either with vehicle or rhIL-11 at a high or low dose and culled on day 6. RESULTS: A high dose of rhIL-11 resulted in a significant reduction in the glomerular expression of TGF-ß1 (0.4 ± 0.1 vs. 2.04 ± 0.4 semiquantitative score, p<0.005), alpha-SMA (0.6 ± 0.2 vs. 1.5 ± 0.3, p<0.01) and fibronectin (0.6 ± 0.1 vs. 1.5 ± 0.1, p<0.02). The periglomerular expression of alpha-SMA and fibronectin was significantly reduced in rats treated with the high dose of rhIL-11 (9.6% ± 2% vs. 92% ± 2.5% of glomeruli, p<0.01; and 26% ± 4.9% vs. 94% ± 1.9% of glomeruli, p<0.005, respectively). There was a slight but insignificant reduction of p-p38 MAPK in IL-11 treated rats. Treatment with low-dose rhIL-11 did not reduce expression of these molecules. CONCLUSION: IL-11 suppresses glomerular expression of TGF-ß1 and extracellular matrix deposition in experimental glomerulonephritis.
Subject(s)
Extracellular Matrix Proteins/metabolism , Glomerulonephritis/prevention & control , Interleukin-11/pharmacology , Kidney Glomerulus/drug effects , Transforming Growth Factor beta1/metabolism , Actins/metabolism , Animals , Autoantibodies , Disease Models, Animal , Fibronectins/metabolism , Glomerulonephritis/immunology , Glomerulonephritis/metabolism , Glomerulonephritis/pathology , Humans , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Male , Phosphorylation , Rats , Rats, Inbred WKY , Recombinant Proteins/pharmacology , Time Factors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Peritoneal fibrosis manifests clinically as membrane failure or encapsulating peritoneal sclerosis (EPS). There are no clinical or biochemical tests to determine the rate of progression of peritoneal fibrosis. CCL18/pulmonary and activation-regulated chemokine (PARC) is profibrotic and stimulates collagen production independent of the effect of transforming growth factor beta. This has not been studied in peritoneal dialysis (PD) patients. MATERIALS AND METHODS: We have prospectively studied 106 patients, free from infection/recent peritonitis. A high concentration of CCL18 was discovered by multiplex antibody arrays and quantified by ELISA. Serum and dialysate levels were examined for their prognostic values. RESULTS: By multiple regression analysis, dialysate CCL18 (6·76 ± 0·66 µg 4 h⻹) correlated with increasing membrane transport status (TS) (P < 0·0001) and total glucose exposure/24 h (P = 0·033). Serum CCL18 correlated with high TS (P = 0·0001) and duration of PD (P = 0·001). After 12 months of follow-up, 57 patients remained on PD while 12 patients were transferred to haemodialysis (HD) and seven developed EPS. Patients who subsequently developed EPS had higher baseline dialysate CCL18 (11·5 ± 3·6 µg 4 h⻹ vs. 5·6 ± 0·82 µg 4 h⻹, P = 0·03) and serum CCL18 (156·9 ± 12·8 ng mL⻹ vs. 124·8 ± 12·2 ng mL⻹, P = 0·02) compared with the stable PD group. CONCLUSION: This is the first report of high levels of CCL18 in the spent dialysate and serum from long-term PD patients. These levels correlated with dysfunction of peritoneal membrane transport status, therefore following CCL18 in a longitudinal study may be of interest.
Subject(s)
Chemokines, CC/blood , Peritoneal Dialysis, Continuous Ambulatory/adverse effects , Peritoneal Fibrosis/diagnosis , Biological Transport , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Prospective Studies , Regression AnalysisABSTRACT
The Wistar Kyoto (WKY) rat is uniquely susceptible to experimentally induced crescentic glomerulonephritis. Two major quantitative trait loci (QTLs) on chromosomes 13 (Crgn1) and 16 (Crgn2) with logarithm of odds >8, as well as five other loci (Crgn3 through 7), largely explain this genetic susceptibility. To understand further the effects of Crgn1 and Crgn2, we generated a double-congenic strain by introgressing these loci from glomerulonephritis-resistant Lewis rats onto the WKY genetic background. Induction of nephrotoxic nephritis in the double-congenic rats (WKY.LCrgn1,2) produced markedly fewer glomerular crescents, reduced macrophage infiltration, and decreased expression of glomerular TNF-alpha and inducible nitric oxide synthase expression compared with control animals. Bone marrow and kidney transplantation studies between parental and WKY.LCrgn1,2 strains, together with in vitro experiments, demonstrated that Crgn1 and Crgn2 contribute exclusively to circulating cell-related glomerular injury by regulating macrophage infiltration and activation. The residual genetic susceptibility to crescentic glomerulonephritis in WKY.LCrgn1,2 rats associated with macrophage activity (especially with enhanced metalloelastase expression) rather than macrophage infiltration. Taken together, these results demonstrate that a genetic influence on macrophage activation, rather than number, determines glomerular damage in immune-mediated glomerulonephritis.
Subject(s)
Genetic Loci/genetics , Genetic Loci/physiology , Glomerular Mesangium/physiopathology , Glomerulonephritis/genetics , Glomerulonephritis/physiopathology , Macrophages/physiology , Animals , Bone Marrow Transplantation , Cells, Cultured , Disease Models, Animal , Genetic Predisposition to Disease/genetics , Glomerular Mesangium/metabolism , Glomerular Mesangium/pathology , Glomerulonephritis/pathology , Kidney Transplantation , Macrophages/pathology , Male , Matrix Metalloproteinase 12/metabolism , Nitric Oxide Synthase Type II/metabolism , Rats , Rats, Inbred Lew , Rats, Inbred WKY , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Antibody-mediated glomerulonephritis, including that resulting from immune complexes, is an important cause of renal failure and is in need of more specific and effective treatment. Binding of antibody or immune complexes to Fc receptors activates intracellular signal transduction pathways, including spleen tyrosine kinase (Syk), leading to the production of inflammatory cytokines. We examined the effect of R788 (fostamatinib disodium), an oral prodrug of the selective Syk inhibitor R406, in nephrotoxic nephritis in Wistar-Kyoto rats. Treatment with R788 reduced proteinuria, tissue injury, glomerular macrophage and CD8+ cell numbers, and renal monocyte chemoattractant protein-1 (MCP-1) and IL-1beta, even when we started treatment after the onset of glomerulonephritis. When we administered R788 from days 4 to 10, glomerular crescents reduced by 100% (P < 0.01) compared with the vehicle group. When we administered R788 treatment from days 7 to 14, established glomerular crescents reversed (reduced by 21%, P < 0.001), and renal function was better than the vehicle group (P < 0.001). In vitro, R406 downregulated MCP-1 production from mesangial cells and macrophages stimulated with aggregated IgG. These results suggest that Syk is an important therapeutic target for the treatment of glomerulonephritis.
Subject(s)
Glomerulonephritis/metabolism , Glomerulonephritis/physiopathology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Oxazines/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Severity of Illness Index , Aminopyridines , Animals , Cells, Cultured , Chemokine CCL2/metabolism , Disease Models, Animal , Glomerulonephritis/pathology , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Mesangial Cells/pathology , Morpholines , Protein-Tyrosine Kinases/metabolism , Pyrimidines , Rats , Rats, Inbred WKY , Signal Transduction/physiology , Syk KinaseABSTRACT
Crescentic glomerulonephritis is an important cause of human kidney failure for which the underlying molecular basis is largely unknown. In previous studies, we mapped several susceptibility loci, Crgn1-Crgn7, for crescentic glomerulonephritis in the Wistar Kyoto (WKY) rat. Here we show by combined congenic, linkage and microarray studies that the activator protein-1 (AP-1) transcription factor JunD is a major determinant of macrophage activity and is associated with glomerulonephritis susceptibility. Introgression of Crgn2 from the nonsusceptible Lewis strain onto the WKY background leads to significant reductions in crescent formation, macrophage infiltration, Fc receptor-mediated macrophage activation and cytokine production. Haplotype analysis restricted the Crgn2 linkage interval to a 430-kb interval containing Jund, which is markedly overexpressed in WKY macrophages and glomeruli. Jund knockdown in rat and human primary macrophages led to significantly reduced macrophage activity and cytokine secretion, indicating conservation of JunD function in macrophage activation in rats and humans and suggesting in vivo inhibition of Jund as a possible new therapeutic strategy for diseases characterized by inflammation and macrophage activation.
Subject(s)
Genetic Predisposition to Disease , Glomerulonephritis/genetics , Macrophage Activation/genetics , Proto-Oncogene Proteins c-jun/physiology , Rats/genetics , Transcription Factor AP-1/physiology , Animals , Animals, Congenic , Chromosome Mapping , Gene Expression , Genetic Linkage , Haplotypes , Humans , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Rats, Inbred Lew , Rats, Inbred WKY , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolismABSTRACT
Interferon-beta (IFN-beta) is a multifunctional cytokine with immunomodulatory properties. We examined the effect of IFN-beta in three separate rat models of glomerular injury and in cultured human glomerular endothelial cells and podocytes. In nephrotoxic nephritis in WKY rats, recombinant rat IFN-beta started either at induction or after establishment of disease significantly reduced 24-h proteinuria by up to 73% and 51%, respectively, but did not affect serum creatinine. There was a slight reduction in numbers of glomerular macrophages, but no difference in glomerular or tubulointerstitial scarring. In Thy-1 nephritis in Lewis rats, IFN-beta started at induction of disease reduced proteinuria by up to 66% with no effect on numbers of glomerular macrophages, but a reduced number of proliferating cells. In puromycin nephropathy in Wistar rats, IFN-beta started at induction of disease reduced proteinuria by up to 93%, but had no effect on glomerular histology. In cultured cells, human IFN-beta-1a had a dramatic effect on barrier properties, increasing electrical resistance across monolayers of either glomerular endothelial cells or podocytes and decreasing trans-monolayer passage of albumin. In conclusion, these results show that IFN-beta reduces proteinuria in three different rat models of glomerular injury and that its anti-proteinuric action may result from direct effects on cells that comprise the glomerular filtration barrier. These data indicate that IFN-beta may have potential as a therapeutic agent in proteinuric renal disease.