RESUMEN
INTRODUCTION: As the immunosuppressive potency of 15-deoxyspergualin (DSG) has been shown in the therapy of renal transplant rejection and Wegener's granulomatosis, the intention of this study was to evaluate the safety of DSG in the therapy of lupus nephritis (LN). METHODS: Patients with histologically proven active LN after prior treatment with at least one immunosuppressant were treated with 0.5 mg/kg normal body weight/day DSG, injected subcutaneously for 14 days, followed by a break of one week. These cycles were repeated to a maximum of nine times. Doses of oral corticosteroids were gradually reduced to 7.5 mg/day or lower by cycle 4. Response was measured according to a predefined decision pattern. The dose of DSG was adjusted depending on the efficacy and side effects. RESULTS: A total of 21 patients were included in this phase-I/II study. After the first DSG injection, one patient was excluded from the study due to renal failure. Five patients dropped out due to adverse events or serious adverse events including fever, leukopenia, oral candidiasis, herpes zoster or pneumonia. Eleven out of 20 patients achieved partial (4) or complete responses (7), 8 were judged as treatment failures and 1 patient was not assessable. Twelve patients completed all nine cycles; in those patients, proteinuria decreased from 5.88 g/day to 3.37 g/day (P = 0.028), Selena-SLEDAI (Safety of Estrogens in Lupus Erythematosus-National Assessment-systemic lupus erythematosus disease activity index) decreased from 17.6 to 11.7. In 13 out of 20 patients, proteinuria decreased by at least 50%; in 7 patients to less than 1 g/day. CONCLUSIONS: Although the number of patients was small, we could demonstrate that DSG provides a tolerably safe treatment for LN. The improvement in proteinuria encourages larger controlled trials. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00709722.
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Guanidinas/administración & dosificación , Inmunosupresores/administración & dosificación , Nefritis Lúpica/tratamiento farmacológico , Adulto , Femenino , Guanidinas/efectos adversos , Humanos , Inmunosupresores/efectos adversos , Masculino , Proyectos Piloto , Adulto JovenRESUMEN
Polyamine compound deoxyspergualin (DSG) is a potent immunosuppressive agent that has been applied clinically for protecting graft rejection and treatment of Wegener's granulomatosis. Though DSG can bind to heat-shock proteins (HSPs) in cells, its mechanism of immunosuppressive action remains unknown. It is widely accepted that extracellular HSPs are capable of stimulating dendritic cells (DC) through cell surface receptors, leading to DC activation and cytokine release. In this study, we examined if DSG analogs could inhibit HSP70-induced DC activation. Bone marrow derived immature mouse DCs and peripheral blood mononuclear cell-derived immature human DCs were generated and incubated with Alexa 488-labeled Hsp70 in the presence of methoxyDSG (Gus-1) that had comparable HSP70-binding affinity to DSG or DSG analog GUS-7, which had much more reduced binding affinity for HSP70. The binding of HSP70 to immature DCs was analyzed by laser microscopy and flow cytometry. HSP70-induced DC activation was assessed by TNF-alpha release by enzyme-linked immunosorbent assay. Binding of Hsp70 to the cell surface of immature DCs was inhibited under the presence of Gus-1, but not under the presence of Gus-7. Immature DCs were activated and released TNF-alpha by the stimulation with HSP70 for 12 hours; however, the HSP70-induced TNF-alpha release was suppressed under the presence of Gus-1, and partially suppressed under the presence of Gus-7. Similar results were observed when immature human DCs were stimulated under the same conditions. Immunosuppressive mechanism of DSG may be explained, at least in part, by the inhibition of extracellular HSP70-DC interaction and HSP70-induced activation of immature DCs.
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Diferenciación Celular/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/inmunología , Guanidinas/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Poliaminas/farmacología , Animales , Antígeno B7-1/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Dendríticas/efectos de los fármacos , Guanidinas/química , Ratones , Ratones Endogámicos C57BL , Unión Proteica/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The therapeutic efficacy of immunosuppressants for treating rapidly progressive glomerulonephritis (RPGN) with crescent formation remains controversial. SCG/Kj mice spontaneously develop RPGN-like symptoms, characteristic of crescentic glomerulonephritis and systemic small vessel vasculitis, associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). We evaluated the "ameliorative", not prophylactic, effects of immunosuppressive agents, deoxyspergualin (DSG), cyclophosphamide (CYC) and prednisolone (PDN), on RPGN in these mice. DSG at intraperitoneal doses of 3 and 6 mg/kg, CYC at an oral dose of 12 mg/kg, or PDN at an intraperitoneal dose of 120 mg/kg was administered once a day for 21 days to female mice "at the onset of hematuria". A set of control SCG/Kj mice received only saline injections. DSG and CYC significantly prolonged survival, improved the proteinuria, hematuria and hyperuremia, and decreased the serum level of myeloperoxidase-ANCA. Moreover, DSG significantly suppressed the formation of crescents in glomeruli. PDN failed to affect any of the parameters. DSG might be useful for inducing remission in crescentic glomerulonephritis involved in RPGN.
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Glomerulonefritis/tratamiento farmacológico , Guanidinas/uso terapéutico , Inmunosupresores/uso terapéutico , Animales , Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Recuento de Células Sanguíneas , Nitrógeno de la Urea Sanguínea , Peso Corporal , Síndrome de Churg-Strauss/genética , Femenino , Glomerulonefritis/genética , Glomerulonefritis/patología , Hematuria/sangre , Hematuria/inducido químicamente , Riñón/patología , Ratones , Ratones Endogámicos , Peroxidasa/metabolismo , Análisis de Supervivencia , UrinálisisRESUMEN
OBJECTIVE: Crescent formation in the renal glomerulus is a typical manifestation of progressive glomerulopathy associated with fatal renal failure; therefore, its prevention is of clinical importance. Little is known about the pathogenic mechanism for crescent formation. This study was undertaken in an attempt to identify the events that are critical for crescent formation in immune complex crescentic glomerulonephritis (CGN) by analyzing a novel mutant strain of mice. METHODS: A spontaneous mutant strain of mice was isolated from the autoimmune-prone strain EOD, which stably develops fatal CGN. The mutant phenotypes were assessed histopathologically, hematologically, and immunologically. The mutation was searched for with positional cloning using microsatellite markers. RESULTS: Compared with wild-type EOD (WT-EOD) mice, mutant EOD (mut-EOD) mice showed marked improvement in CGN in conjunction with an improvement in spontaneous mortality. In WT-EOD mice, an inverse correlation between blood urea nitrogen concentration and blood platelet count and massive accumulation of platelets in the glomerulus were evident, suggesting that an accumulation of platelets in the glomerulus contributes to the progression of CGN. The mutant platelets showed an abnormal aggregation in response to collagen and thrombin, associated with a bleeding tendency in mut-EOD mice. Genetic analysis revealed a deleterious mutation in the cappuccino gene (cno), which encodes a protein that belongs to a complex called the biogenesis of lysosome-related organelle complex 1 and is profoundly involved in platelet function. Morphologic examination revealed a partial defect in dense body formation in the delta-granule of platelets. CONCLUSION: The present findings suggest that platelet functions have a critical role in crescent formation in autoimmune GN.
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Plaquetas/fisiología , Glomerulonefritis/genética , Proteínas de Transporte Vesicular/genética , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/genética , Recuento de Células Sanguíneas , Nitrógeno de la Urea Sanguínea , Cartilla de ADN , Glomerulonefritis/sangre , Glomerulonefritis/inmunología , Glomerulonefritis/patología , Immunoblotting , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , FenotipoRESUMEN
BACKGROUND: Here we compare the efficacy of cyclophosphamide (CYC) for treatment of crescentic nephritis (CGN) with the newer immunosuppressants 15-deoxyspergualin (DSG) and mycophenolate mofetil (MMF) in SCG/Kj mice, an inbred mouse strain that spontaneously develops CGN, systemic necrotizing vasculitis and antineutrophil cytoplasmic antibodies (ANCAs). METHODS: Mice were randomly assigned to intraperitoneal treatment with either DSG (2 mg/kg/day), CYC (50 mg/kg/week), MMF (60 or 100 mg/kg/day) or vehicle (VEH, dextrose 5% 0.3 ml/day) beginning at the 10th week of life. ANCA, blood urea nitrogen (BUN) and proteinuria were determined in all animals regularly, and survival was calculated. Renal histology was obtained in the 18th week of life in the MMF- or VEH-treated groups and in the 24th week in DSG- or CYC-treated animals. RESULTS: Mean survival in VEH-treated animals was 123 days. At that point, survival was 100% in the CYC- or DSG-treated animals (P<0.001). Survival in the MMF group (pooled data) was not significantly different from the VEH-treated animals [MMF, 117 days (95% CI 108-127)]. BUN (18th week, CYC 43+/-9 mg/dl and DSG 36+/-6 mg/dl vs VEH 73+/-28 mg/dl, P<0.001, MMF 66+/-26 mg/dl), 24 h proteinuria (18th week, CYC 0.4+/-0.2 mg and DSG 0.7+/-0.6 mg vs VEH 2.7+/-3 mg, P<0.001, MMF 2.2+/-3 mg) crescent formation (18th week, VEH 42+/-9%, MMF 39+/-11%; CYC 5+/-2% and DSG 22+/-7% vs VEH, P<0.05), glomerular immune complex deposition, and ANCA formation were significantly improved in CYC- and DSG- but not in MMF-treated animals when compared with controls. CONCLUSION: DSG and CYC, but not MMF, prolong life, limit renal damage and prevent autoantibody formation in SCG/Kj mice.
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Ciclofosfamida/farmacología , Guanidinas/farmacología , Inmunosupresores/farmacología , Ácido Micofenólico/análogos & derivados , Nefritis/tratamiento farmacológico , Nefritis/mortalidad , Animales , Biopsia con Aguja , Modelos Animales de Enfermedad , Inmunohistoquímica , Terapia de Inmunosupresión/métodos , Ratones , Ratones Endogámicos , Ácido Micofenólico/farmacología , Nefritis/inmunología , Nefritis/patología , Distribución Aleatoria , Factores de Riesgo , Sensibilidad y Especificidad , Tasa de SupervivenciaRESUMEN
BACKGROUND: Myeloperoxidase-specific anti-neutrophil cytoplasmic auto-antibody (MPO-ANCA) has been a useful diagnostic marker in systemic vasculitis with crescentic glomerulonephritis (CrGN). It is highly suspected that the antigenic enzyme MPO released from activated neutrophils is involved in these lesions. We evaluated the relationship between neutrophil functions including peripheral neutrophil counts and renal lesions in SCG/Kj mice as a model of ANCA-associated CrGN and vasculitis. METHODS: Peripheral neutrophil counts, the plasma levels of MPO-ANCA and tumour necrosis factor alpha (TNF-alpha) were measured. The capacity of MPO release and superoxide generation were evaluated as neutrophil activity. The renal lesions were estimated by grade of proteinuria, histopathological lesion, such as glomerular neutrophil infiltration and active or chronic renal injury scores with crescent formation. RESULTS: MPO-ANCA and TNF-alpha levels were higher than those of normal mice C57BL/6 even before overt proteinuria; subsequently, peripheral neutrophils increased. In the phase of nephritis with low grade proteinuria, the spontaneous release of MPO from peripheral neutrophils increased, while superoxide generation increased before spontaneous MPO release occurred. In addition, the renal lesion in histological observations was aggravated with ageing and the glomerular neutrophil infiltration was positively correlated with MPO-ANCA levels, as well as with histological indices of nephritis, active renal injury score; in particular, crescent formation was correlated with spontaneous MPO release. In contrast, superoxide generation was negatively correlated with the severity of this lesion during the progression. CONCLUSIONS: These findings indicate that neutrophils are activated and contribute to the development of the active crescentic lesion in SCG/Kj mice.
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Glomerulonefritis/inmunología , Neutrófilos/fisiología , Animales , Femenino , Glomerulonefritis/sangre , Glomerulonefritis/patología , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice spontaneously develop crescentic glomerulonephritis (CGN), systemic vasculitis, and perinuclear ANCA (pANCA), and have been suggested as an animal model for human antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AASV). Since no systematic serologic, immunohistologic, or structural evaluation had been performed thus far, we reinvestigated the development of ANCA and CGN in these mice. METHODS: SCG/Kj mice were subjected to serologic and urinary analysis, as well as histologic evaluation of the kidneys by standard light, immunofluorescence, and electron microscopy at regular intervals during the course of the disease. RESULTS: Perinuclear ANCA developed as early as the 6th week of life, increasing both in frequency and titer in up to 100% of animals at week 20. Crescent formation began at week 10 and peaked at week 16, maximally affecting 57% of glomeruli. Crescent formation was initiated by "activated" podocytes that formed cell bridges between tuft and Bowman's capsule. The typical picture of a diffuse immune complex nephritis was found in all animals as early as 8 weeks. Fluorescence intensity increased with age and became strongly positive for immunoglobulin (Ig)A, IgM, IgG, and C3 in the mesangium and along the peripheral capillary loops. CONCLUSION: Although ANCAs were found in the majority of animals, the massive presence of glomerular immune deposits differed from the pauci-immune pattern found in human AASV, making this model not completely representative for human ANCA-associated CGN. However, the spontaneous and concomitant development of pANCA, small vessel vasculitis, and CGN raises the opportunity to analyze pathogenetic links between these disease manifestations in vivo.
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Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Complemento C3/metabolismo , Modelos Animales de Enfermedad , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Inmunoglobulinas/metabolismo , Ratones Endogámicos , Animales , Capilares/patología , Femenino , Glomerulonefritis/complicaciones , Ratones , Necrosis , Vasculitis/etiología , Vasculitis/patologíaRESUMEN
BACKGROUND: Spontaneous crescentic glomerulonephritis (SCG)/Kj mice are a candidate for suitable animal model of human pauci-immune crescentic glomerulonephritis (GN). In the present study, we used renal biopsy technique and analyzed time sequence correlations among crescent formation and glomerular neutrophil infiltration in SCG/Kj mice. METHODS: We observed the progress of GN in SCG/Kj mice according to the urinary abnormalities, and performed the serial renal biopsies. The kinetics of histopathology and glomerular neutrophil influx corresponding disease stage were examined by enzyme-histochemistry and immunohistochemistry. RESULTS: We divided natural course of GN into three periods in view of urinalysis: a proteinuria-negative/hematuria-negative (P- H-) period, followed by a proteinuria-positive/hematuria-negative (P+ H-) period, and finally a proteinuria-positive/hematuria-positive (P+ H+) period. Endocapillary proliferation phase existed in P+ H- period, whereas crescent formation occurred and extended in P+ H+ period. In P+ H- period, prominent glomerular neutrophil infiltration was observed, while these numbers decreased with the progression of crescent formation. CONCLUSION: These observations suggest that there is a good correlation between urinalysis and histopathological events of SCG/Kj mice, and that endocapillary proliferation, which contains neutrophil infiltration, may contribute to the subsequent crescent formation in these mice.