RESUMEN
Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus; in LN class IV morphologic lesions may be similar to the lesions in primary membranoproliferative glomerulonephritis (MPGN). The aim of the study was to compare the counts of tryptase-positive and chymase-positive mast cells between LN class IV and MPGN. The material consisted of 61 renal biopsies: 32 with lupus nephritis class IV, and 29 with membranoproliferative glomerulonephritis. Chymase- and tryptase-positive cells were stained by immunohistochemistry and subsequently counted. The mean count of chymase-positive mast cells was 21.94 for the whole group, 12.66 for LN class IV and 32.18 for MPGN. The mean count of tryptase-positive cells was 34.94 hpf for the entire group, 22.98 for LN class IV and 48. 13 for MPGN. The differences between lupus nephritis and membranoproliferative glomerulonephritis were significant both for chymase- and tryptase-positive cells. Both chymase-positive MC counts and tryptase-positive MC counts correlated with relative interstitial volume (RIV) (R=0.35 and R=0.28, respectively) and with creatinine level (R=0.35 and R=0.43, respectively). There was also a significant correlation between age, creatinine level and RIV (R=0.28 and R=0.26, respectively).
Asunto(s)
Glomerulonefritis Membranoproliferativa/patología , Nefritis Lúpica/patología , Mastocitos/patología , Adulto , Biomarcadores/análisis , Biopsia , Recuento de Células , Quimasas/análisis , Diagnóstico Diferencial , Femenino , Glomerulonefritis Membranoproliferativa/enzimología , Humanos , Inmunohistoquímica , Nefritis Lúpica/enzimología , Masculino , Mastocitos/enzimología , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Índice de Severidad de la Enfermedad , Triptasas/análisis , Adulto JovenRESUMEN
BACKGROUND: Chronic progressive mesangioproliferative nephropathy represents a major cause of end-stage renal disease worldwide. Until now, effective approaches to stop or even slow its progression are limited. We tested the effects of an inhibitor of PDGF receptor, abl and c-kit tyrosine kinases, Imatinib, in a chronic progressive model of mesangioproliferative glomerulosclerosis. METHODS: Anti-thy1 glomerulosclerosis was induced by injection of anti-thy1 antibody into uninephrectomized Wistar rats. One week after disease induction, according to the degree of proteinuria, animals were stratified and assigned to chronic glomerulosclerosis (cGS) and cGS plus Imatinib (10 mg/kg body weight/day). In week 20, renoprotective actions of Imatinib were analyzed by a set of functional, histological and molecular biological parameters. RESULTS: Untreated cGS rats showed elevation of systolic blood pressure and marked progression in proteinuria, renal fibrosis, cell infiltration, cell proliferation and function lost. Administration of Imatinib went along significantly with lower systolic blood pressure (-10 mmHg) and proteinuria (-33%). Imatinib administration was paralled by significant reductions in tubulointerstitial accumulation of matrix proteins (-44%), collagen I deposition (-86%), expression of TGF-beta1 (-30%), production of fibronectin (-23%), myofibroblast differentiation (-87%), macrophage infiltration (-36%) and cell proliferation (-45%), respectively. In comparison with untreated cGS animals, Imatinib therapy lowered also blood creatinine (-41%) and blood urea concentrations (-36%) and improved creatinine clearance (+25%). Glomerular fibrotic changes were lowered moderately by Imatinib. CONCLUSIONS: Therapy with Imatinib limits the progressive course of chronic anti-thy1 glomerulosclerosis towards tubulointerstitial fibrosis and renal insufficiency. This was paralleled by direct and indirect sign of TGF-ß1 and PDGF inhibition. The findings suggest that the pharmacological principal of inhibition of tyrosine kinases with drugs such as Imatinib might serve as approach for limiting progression of human mesangioproliferative glomerulosclerosis.
Asunto(s)
Benzamidas/uso terapéutico , Glomerulonefritis Membranoproliferativa/tratamiento farmacológico , Glomerulonefritis Membranoproliferativa/enzimología , Piperazinas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Animales , Enfermedad Crónica , Progresión de la Enfermedad , Glomerulonefritis Membranoproliferativa/inducido químicamente , Glomerulonefritis Membranoproliferativa/inmunología , Mesilato de Imatinib , Masculino , Inhibidores de Proteínas Quinasas/uso terapéutico , Ratas , Ratas Wistar , Antígenos Thy-1/inmunología , Resultado del TratamientoRESUMEN
Renal microangiopathies and membranoproliferative GN (MPGN) can manifest similar clinical presentations and histology, suggesting the possibility of a common underlying mechanism in some cases. Here, we performed homozygosity mapping and whole exome sequencing in a Turkish consanguineous family and identified DGKE gene variants as the cause of a membranoproliferative-like glomerular microangiopathy. Furthermore, we identified two additional DGKE variants in a cohort of 142 unrelated patients diagnosed with membranoproliferative GN. This gene encodes the diacylglycerol kinase DGKε, which is an intracellular lipid kinase that phosphorylates diacylglycerol to phosphatidic acid. Immunofluorescence confocal microscopy demonstrated that mouse and rat Dgkε colocalizes with the podocyte marker WT1 but not with the endothelial marker CD31. Patch-clamp experiments in human embryonic kidney (HEK293) cells showed that DGKε variants affect the intracellular concentration of diacylglycerol. Taken together, these results not only identify a genetic cause of a glomerular microangiopathy but also suggest that the phosphatidylinositol cycle, which requires DGKE, is critical to the normal function of podocytes.
Asunto(s)
Diacilglicerol Quinasa/genética , Glomerulonefritis Membranoproliferativa/enzimología , Glomerulonefritis Membranoproliferativa/genética , Enfermedades Renales/enzimología , Enfermedades Renales/genética , Mutación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Estudios de Cohortes , Consanguinidad , ADN/genética , Diacilglicerol Quinasa/metabolismo , Diagnóstico Diferencial , Diglicéridos/metabolismo , Femenino , Variación Genética , Glomerulonefritis Membranoproliferativa/patología , Células HEK293 , Humanos , Enfermedades Renales/patología , Glomérulos Renales/enzimología , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Podocitos/metabolismo , Polimorfismo de Nucleótido Simple , Ratas , Homología de Secuencia de Aminoácido , TurquíaRESUMEN
AIM: Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy where therapy targeting the ß isoform of this enzyme has been examined. However, PKC-ß is also increased in various forms of human glomerulonephritis, including IgA nephropathy. Accordingly, we sought to examine the effects of PKC-ß inhibition in the Thy1.1 model of mesangial proliferative glomerulonephritis. METHODS: Following administration of monoclonal OX-7, anti-rat Thy-1.1 antibody, Male Wistar rats were randomized to receive either the PKC-ß inhibitor, ruboxistaurin (10 mg/kg per day in chow) or vehicle. Animals were then examined 6 days later. RESULTS: PKC-ß inhibition was associated with reductions in mesangial cellularity and extracellular matrix deposition. Proteinuria was, however, unaffected. In vitro, PKC-ß inhibition showed modest, dose-dependent reductions in mesangial cell (3) H-thymidine and (3) H-proline incorporations, indices of cell proliferation and collagen synthesis, respectively. CONCLUSION: The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by PKC-ß inhibition suggests the potential clinical utility of this approach as a therapeutic strategy in non-diabetic glomerular disease.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Glomerulonefritis Membranoproliferativa/tratamiento farmacológico , Indoles/farmacología , Maleimidas/farmacología , Células Mesangiales/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Anticuerpos Monoclonales , Línea Celular , Colágeno/biosíntesis , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Glomerulonefritis Membranoproliferativa/enzimología , Glomerulonefritis Membranoproliferativa/inmunología , Glomerulonefritis Membranoproliferativa/patología , Inmunoglobulina G , Masculino , Células Mesangiales/enzimología , Células Mesangiales/inmunología , Células Mesangiales/patología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Proteinuria/tratamiento farmacológico , Proteinuria/enzimología , Ratas , Ratas Wistar , Antígenos Thy-1/inmunologíaRESUMEN
BACKGROUND: Deposition of chromatin-IgG complexes within glomerular membranes is a key event in the pathogenesis of lupus nephritis. We recently reported an acquired loss of renal Dnase1 expression linked to transformation from mild to severe membranoproliferative lupus nephritis in (NZBxNZW)F1 mice. As this may represent a basic mechanism in the progression of lupus nephritis, several aspects of Dnase1 expression in lupus nephritis were analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Total nuclease activity and Dnase1 expression and activity was evaluated using in situ and in vitro analyses of kidneys and sera from (NZBxNZW)F1 mice of different ages, and from age-matched healthy controls. Immunofluorescence staining for Dnase1 was performed on kidney biopsies from (NZBxNZW)F1 mice as well as from human SLE patients and controls. Reduced serum Dnase1 activity was observed in both mesangial and end-stage lupus nephritis. A selective reduction in renal Dnase1 activity was seen in mice with massive deposition of chromatin-containing immune complexes in glomerular capillary walls. Mice with mild mesangial nephritis showed normal renal Dnase1 activity. Similar differences were seen when comparing human kidneys with severe and mild lupus nephritis. Dnase1 was diffusely expressed within the kidney in normal and mildly affected kidneys, whereas upon progression towards end-stage renal disease, Dnase1 was down-regulated in all renal compartments. This demonstrates that the changes associated with development of severe nephritis in the murine model are also relevant to human lupus nephritis. CONCLUSIONS/SIGNIFICANCE: Reduction in renal Dnase1 expression and activity is limited to mice and SLE patients with signs of membranoproliferative nephritis, and may be a critical event in the development of severe forms of lupus nephritis. Reduced Dnase1 activity reflects loss in the expression of the protein and not inhibition of enzyme activity.
Asunto(s)
Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Regulación Enzimológica de la Expresión Génica , Glomerulonefritis Membranoproliferativa/enzimología , Riñón/enzimología , Nefritis Lúpica/enzimología , Animales , Biopsia , ADN/metabolismo , Desoxirribonucleasa I/sangre , Desoxirribonucleasa I/deficiencia , Progresión de la Enfermedad , Electroforesis , Femenino , Técnica del Anticuerpo Fluorescente , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/metabolismo , Humanos , Riñón/metabolismo , Lupus Eritematoso Sistémico/enzimología , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/genética , Nefritis Lúpica/metabolismo , Ratones , Transporte de ProteínasRESUMEN
Immunomodulatory enzyme indoleamine 2, 3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. Via catalysing tryptophan degradation, IDO suppresses adaptive T cell-mediated immunity and plays an important role in various forms of immune tolerance. Its role in T helper type 1 (Th1)-directed, cell-mediated crescentic glomerulonephritis (GN) is still unclear. Therefore, we investigated the activity and role of IDO in crescentic GN using a model of nephrotoxic serum nephritis (NTN), and IDO activity was inhibited by 1-methyl-tryptophan (1-MT) in vivo. Our results showed that activity of IDO, as determined by high performance liquid chromatography analysis of the kynurenine/tryptophan ratio, was increased markedly in the serum and renal tissue of NTN mice, and immunohistochemistry revealed that expression of IDO was up-regulated significantly in glomeruli and renal tubular epithelial cells during NTN. Treatment with 1-MT resulted in significantly exacerbated kidney disease with increased glomerular crescent formation, accumulation of CD4(+)T cells and macrophages in renal tissue, and augmented renal injury compared with phosphate-buffered saline-treated NTN mice, which was associated with enhanced Th1 responses and intrarenal cellular proliferation. These findings suggest that the development of NTN was regulated negatively by increased IDO activity, and IDO might play an important role in the pathogenesis of crescentic GN.
Asunto(s)
Glomerulonefritis Membranoproliferativa/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Glomérulos Renales/enzimología , Túbulos Renales/enzimología , Triptófano/análogos & derivados , Animales , Linfocitos T CD4-Positivos/inmunología , Cromatografía Líquida de Alta Presión , Glomerulonefritis Membranoproliferativa/inmunología , Glomerulonefritis Membranoproliferativa/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/análisis , Glomérulos Renales/inmunología , Túbulos Renales/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Triptófano/farmacologíaRESUMEN
Mast cell-derived chymase is an angiotensin II-forming enzyme that appears to be involved in tubulointerstitial fibrosis in the kidneys. Previous studies have shown that the level of chymase increases in grafted kidneys after rejection and in adult patients with diabetic nephropathy. However, the significance of chymase in children with renal diseases has not been investigated. Using immunohistochemistry, we have investigated chymase expression in biopsy samples of renal tissue from 104 children with kidney diseases, including rapidly progressive crescentic glomerulonephritis (n = 3), diabetic nephropathy (n = 2), allografted kidney (n = 3), membranoproliferative glomerulonephritis (n = 6), immunoglobulin A nephropathy (n = 33) and Henoch-Schönlein purpura nephritis (n = 23). Increased numbers of chymase-positive mast cells were observed in the renal cortex of all three patients with crescentic glomerulonephritis (mean 26.0/mm(2); range 19.3-36.8/mm(2)). Chymase-positive cells were also observed in the renal biopsy of an allografted kidney and in those from children with diabetic nephropathy. The mean number of chymase-positive cells in renal tissue samples characterized by each renal disease was significantly correlated with the mean intensity of the interstitial fibrosis in that same tissue sample (Spearman's rank correlation test p = 0.0013; rank correlation coefficient 0.84). These findings suggest that mast cell-derived chymase plays an important role in juvenile crescentic glomerulonephritis.
Asunto(s)
Quimasas/metabolismo , Glomerulonefritis por IGA/enzimología , Riñón/enzimología , Mastocitos/enzimología , Biopsia , Niño , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/patología , Femenino , Fibrosis , Tasa de Filtración Glomerular , Glomerulonefritis por IGA/patología , Glomerulonefritis por IGA/fisiopatología , Glomerulonefritis Membranoproliferativa/enzimología , Glomerulonefritis Membranoproliferativa/patología , Humanos , Vasculitis por IgA/complicaciones , Riñón/patología , Trasplante de Riñón/patología , Masculino , Mastocitos/patología , Nefritis/enzimología , Nefritis/etiología , Nefritis/patologíaRESUMEN
Several studies have demonstrated the effectiveness of arginine analog nitric oxide synthase (NOS) inhibitor therapy in preventing and treating murine lupus nephritis. However, MRL/MpJ-FAS (MRL/lpr) mice lacking a functional NOS2 (inducible NOS [iNOS]) gene (NOS2) develop proliferative glomerulonephritis in a fashion similar to their wild-type (wt) littermates. This finding suggests that the effect of arginine analog NOS inhibitors is through a non-iNOS-mediated mechanism. This study was designed to address this hypothesis.NOS2 mice were given either vehicle or a NOS inhibitor (SD-3651) to determine if pharmacological NOS inhibition prevented glomerulonephritis, using wt mice as positive controls. Urine was collected fortnightly to measure albumin. At the time of full disease expression in wt mice, all mice were killed, and renal tissue was examined for light, immunofluorescence, and electron microscopic evidence of disease. Serum was analyzed for anti-double-stranded DNA antibody production.NOS2 mice had higher serum anti-double-stranded DNA antibody antibody levels than those of wt mice. SD-3651 therapy reduced proteinuria, glomerular immunoglobulin G deposition, and electron microscopic evidence of podocytopathy and endothelial cell swelling without affecting proliferative lesions by light microscopy.These studies confirm that genetic iNOS deficiency alone is insufficient to prevent proliferative glomerulonephritis and suggest that iNOS activity may inhibit autoantibody production. These results also suggest that SD-3651 therapy acts via a non-iNOS-mediated mechanism to prevent endothelial cell and podocyte pathology. Studies that elucidate this mechanism could provide a useful drug target for the treatment of nephritis.
Asunto(s)
Lisina/análogos & derivados , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/deficiencia , Proteinuria/prevención & control , Animales , Anticuerpos Antinucleares/sangre , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Glomerulonefritis Membranoproliferativa/enzimología , Glomerulonefritis Membranoproliferativa/inmunología , Glomerulonefritis Membranoproliferativa/prevención & control , Nefritis Lúpica/enzimología , Nefritis Lúpica/inmunología , Nefritis Lúpica/prevención & control , Lisina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Proteinuria/enzimología , Proteinuria/inmunologíaRESUMEN
The protein kinase C (PKC) family consists of 13 members categorized as conventional or novel depending on whether diacylglycerol, calcium, or phosphatidylserine is required for activation. High glucose leads to activation of different forms of PKC across tissue types, thus determining the kind of diabetes-induced organ damage. PKC beta was reported to have a positive role in B-lymphocyte activity through activation of NF-kB, leading to various immune disorders. We examined renal expression of two PKC isoforms alpha and beta in renal biopsies of patients with diabetic nephropathy, lupus nephritis (LN) (Class 3-4), and mesangioproliferative glomerulonephritis (MPGN) to explore the role of each isoform in different glomerular diseases. PKC alpha and beta gene expression was studied by quantitative real-time reverse transcription-PCR in 20 patients with type 2 diabetes and proteinuria (serum creatinine 2.04 +/- 0.85 mg/dl, 24-h urinary protein 3.61 +/- 1.75 g, eGFR 37.85 +/- 17.89 ml/min/1.73 m2), 20 patients with proliferative LN (serum creatinine 1.67 +/- 1.50 mg/dl, 24-h urinary protein 4.46 +/- 5.01 g, eGFR 69.62 +/- 40.93 ml/min/1.73 m2), and 20 patients with MPGN (serum creatinine 3.32 +/- 2.79 mg/dl, 24-h urinary protein 4.65 +/- 4.11 g, eGFR 32.62 +/- 29.56 ml/min/1.73 m2). Normal tissues from the normal pole of four kidneys removed because of renal tumor served as controls. PKC á gene expression was significantly increased in diabetic kidneys compared to LN and MPGN (316.95 +/- 152.94 microg/ml vs. 185.97 +/- 32.13 and 195.46 +/- 46.45 microg/ml, p < 0.05). PKC â gene expression was significantly increased in the LN and MPGN groups compared to the diabetic nephropathy group (41.01 +/- 14.03 and 39.93 +/- 16.41 microg/ml, respectively, vs. 18.20 +/- 4.91 microg/ml, p < 0.05). Significant correlation was noted between the PKC alpha gene concentrations and proteinuria in diabetic patients. Renal expression of PKC alpha and beta genes in control tissues were significantly lower compared to diabetic kidneys, LN, and MPGN groups (32.31 +/- 0.36 and 4.67 +/- 2.41 microg/ml, respectively, p < 0.001). The study revealed enhanced renal gene expression of both PKC isoforms alpha and beta in diabetic kidney tissues, LN, and MPGN, but in different patterns. PKC alpha gene expression was significantly increased in diabetic patients with chronic kidney disease. The increased expression of the PKC beta gene in LN and MPGN highlights its role in regulation of the immune system. This may represent potential therapeutic targets for prevention of progressive kidney injury in diabetic and proliferative glomerular diseases.
Asunto(s)
Nefropatías Diabéticas/enzimología , Perfilación de la Expresión Génica , Glomerulonefritis Membranoproliferativa/enzimología , Isoenzimas/genética , Nefritis Lúpica/enzimología , Proteína Quinasa C/genética , Adulto , Secuencia de Bases , Cartilla de ADN , Nefropatías Diabéticas/genética , Femenino , Glomerulonefritis Membranoproliferativa/genética , Humanos , Nefritis Lúpica/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: Crescentic glomerulonephritis (crGN) is a frequent and life-threatening manifestation of antineutrophil cytoplasmatic antibody-associated vasculitis. Up-regulation of proinflammatory cytokines contributes to renal damage by activation of p38 mitogen-activated protein kinases (MAPKs). However, it is unclear which of the four p38MAPK isoforms are expressed, activated and hence of major importance in crGN. METHODS: Kidney biopsies of patients with antineutrophil cytoplasmatic antibody-positive crGN and control samples were investigated for the expression and phosphorylation of p38MAPK isoforms and downstream target kinase MAPKAP2 by immunohistochemistry. Expression and functional activation of p38MAPK isoforms by TNF was also assessed in a human podocyte cell line by reverse transcription-polymerase chain reaction, immunoblotting and kinase array. RESULTS: Strong expression of p38MAPKalpha, beta and gamma isoforms was found in glomerular podocytes and crescents. Infiltrating leucocytes showed predominant p38MAPKalpha expression. Activation of p38MAPK and its downstream mediator MAPKAP2 was found in crGN confined to glomerular podocytes, crescents and inflammatory infiltrates. Interestingly, corticosteroid treatment before kidney biopsy diminished p38MAPK activation in crGN. Activated p38MAPK co-localised with alpha, beta and gamma isoforms in podocytes and crescents, while leucocytes showed mainly p38MAPKalpha activation. In a human podocyte cell line mRNA and protein of all four p38MAPK isoforms was expressed but only p38MAPKalpha was activated upon challenge with TNF. CONCLUSIONS: This study shows selective p38MAPK isoform expression and activation in crGN. Podocytes and podocyte-induce crescent formation is the main source of p38MAPK activation in crGN. TNF is a potent and selective activator of the alpha-isoform in podocytes, which therefore appears as a main contributor to proinflammatory signalling in the glomerulum of crGN.
Asunto(s)
Glomerulonefritis Membranoproliferativa/enzimología , Glomérulos Renales/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Biopsia , Western Blotting/métodos , Estudios de Casos y Controles , Células Cultivadas , Activación Enzimática , Glomerulonefritis Membranoproliferativa/inmunología , Granulomatosis con Poliangitis/inmunología , Granulomatosis con Poliangitis/metabolismo , Humanos , Inmunohistoquímica , Isoenzimas/análisis , Isoenzimas/metabolismo , Leucocitos/enzimología , Macrófagos/enzimología , Proteína Quinasa 14 Activada por Mitógenos/análisis , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Fosforilación , Podocitos/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
FTY720 is a novel immune modulator whose primary action is blood lymphocyte depletion through interaction with sphingosine-1-phosphate (S1P) receptors. The present study analyzes the effect of FTY720 on both the early mesangial cell injury and the subsequent matrix expansion phase of experimental mesangioproliferative glomerulonephritis. Disease was induced by injection of OX-7 anti-thy1 antibody into male Wistar rats. In both protocols, FTY720 administration (0.3 mg/kg body wt) resulted in a selective and very marked reduction in blood lymphocyte count. In the injury experiment, the S1P receptor modulator was given starting 5 days before and continued until 1 day after antibody injection. FTY720 did not significantly affect the degree of anti-thy1-induced mesangial cell lysis and glomerular-inducible nitric oxide production. In the matrix expansion experiment, FTY720 treatment was started 1 day after antibody injection and continued until day 7. In this protocol, the S1P modulator reduced proteinuria, histological matrix expansion, and glomerular protein expression of TGF-beta(1), fibronectin, and PAI-1. Glomerular collagen III staining intensity was decreased. FTY720 reduced markedly glomerular lymphocyte number per cross section and to a lesser degree macrophage infiltration. In conclusion, FTY720 significantly limits TGF-beta(1) overexpression and matrix protein expression following induction of acute anti-thy glomerulonephritis, involving reductions in blood and glomerular lymphocyte numbers. The results suggest that lymphocytes actively contribute to matrix expansion in experimental mesangioproliferative glomerulonephritis. Our study expands on findings on FTY720's beneficial effects on tubulointerstitial and functional disease progression previously reported in anti-thy1-induced chronic glomerulosclerosis.
Asunto(s)
Anticuerpos Bloqueadores/toxicidad , Matriz Extracelular/patología , Glomerulonefritis Membranoproliferativa/tratamiento farmacológico , Glomerulonefritis Membranoproliferativa/patología , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/efectos de los fármacos , Esfingosina/análogos & derivados , Antígenos Thy-1/inmunología , Animales , Presión Sanguínea/fisiología , Peso Corporal/efectos de los fármacos , Colágeno Tipo III/biosíntesis , Matriz Extracelular/enzimología , Fibronectinas/biosíntesis , Clorhidrato de Fingolimod , Glomerulonefritis Membranoproliferativa/enzimología , Frecuencia Cardíaca/fisiología , Inmunohistoquímica , Glomérulos Renales/patología , Recuento de Leucocitos , Lipopolisacáridos/farmacología , Masculino , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Proteinuria/prevención & control , Ratas , Ratas Wistar , Esfingosina/farmacología , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
We investigated the effects of paraoxonase (PON1) 192 polymorphism on serum PON1 activity and the impact of phenotypic expression on the risk and prognosis of Turkish children with membranoproliferative glomerulonephritis (MPGN). Eighteen children with biopsy-proven Type I MPGN (10 boys, 8 girls) and age-matched 53 healthy controls were included in the study. PCR (polymerase chain reaction), RFLP (restriction fragment length polymorphism) and agarose gel electrophoresis techniques were used to determine the PON1 192 genotype. PON1 activity was measured by spectrophotometric assay of p-nitrophenol production following addition of paraoxon. We found that PON1 192 genotype distribution (AA, AB, BB) in MPGN patients were 61.1%, 22.3%, 16.6% and 15.1%, 35.8%, 49.1% in controls, respectively. The frequency of AA genotypes was significantly higher in the MPGN group (0.611) compared with the healthy controls (0.151) (p < 0.001). Although the serum PON1 activity was lower in MPGN patients (103.3 +/- 55.2 U/l) than the healthy controls (130.9 +/- 71.2 U/mol), the difference was not statistically significant (p = 0.0563). In the genotypes of patients and controls classified according to PON1 A/B polymorphism; serum PON1 activities were significantly increased (p < 0.001, ANOVA) in the order of PON1 AA, AB and BB in both MPGN patients (82.4, 91.7 and 173.6 U/l) and healthy controls (85.9, 119.9 and 193.1 U/l), respectively. There was a significant relationship between the poor prognosis and having AA genotype and low PON1 activity. Of the 8 patients with poor prognosis, 7 had genotype AA and the remaining one was AB heterozygote. Our results suggest that homozygosity for the A allele might have an important role on the risk for developing MPGN and may also be associated with the poor prognosis of disease. In conclusion, we suggest that the PON1 activities are affected by PON1 genetic variability in Turkish patients with MPGN.
Asunto(s)
Arildialquilfosfatasa/sangre , Arildialquilfosfatasa/genética , Glomerulonefritis Membranoproliferativa/enzimología , Glomerulonefritis Membranoproliferativa/genética , Polimorfismo de Longitud del Fragmento de Restricción , Adolescente , Arginina/genética , Niño , Preescolar , Femenino , Frecuencia de los Genes , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Genotipo , Glutamina/genética , Humanos , Masculino , Valores de Referencia , Medición de Riesgo , Factores de Riesgo , TurquíaRESUMEN
AIM: To study expression of topoisomerases (TI) I and II alpha (DNA-bound enzymes involved in transcription and replication) in renal tissue as markers of activity and prognosis of glomerulonephritis (GN) decisive for choice of immunodepressive therapy. MATERIAL AND METHODS: TI expression was studied immunohistochemically in renal biopsies from 177 patients with different morphological variants of GN and in the samples of unaffected kidney tissue removed in 12 patients for local tumors. RESULTS: There are definite differences between proliferative and non-proliferative GN variants--elevation of TI levels and monocytic infiltration in proliferative GN. Focal-segmental glomerulosclerosis is characterized by a high TI II alpha level in mesangial cells and monocytic infiltration of the glomeruli which are typical for inflammation. A statistical relationship between TI levels in mesangial cells and glomerular epithelium suggests a pathogenetic relation between these links of the pathological process. Molecular markers of activation and proliferation of cells and direct inductors of the inflammatory process (cells of monocytic infiltrate) closely correlated with the activity index--an integral indicator of inflammatory activity, as well as with the integral indicator of sclerotic processes in renal tissue--sclerosis index. Monocytic infiltration in the interstitium correlated both with morphological manifestations of activity, progression of nephritis and their clinical equivalents. In high TI expression GN resistance to immunodepressive therapy rose. To overcome the resistance, immunodepressive therapy must be more active--large doses and duration of treatment. In patients with lupus nephritis and mesangiocapillary GN renal prognosis was worse in the presence of high TI expression in mesangial cells and epithelium of the renal canaliculi. CONCLUSION: The authors are the first to demonstrate TI expression in renal tissue of GN patients, correlation of its level with activity of renal process as well as its role in prediction of response to treatment and the rate of renal failure progression. It is suggested that high TI expression entails a progressive course of GN.
Asunto(s)
ADN-Topoisomerasas/biosíntesis , Glomerulonefritis/enzimología , Riñón/enzimología , Biomarcadores/análisis , Biopsia , ADN-Topoisomerasas/análisis , Glomerulonefritis/diagnóstico , Glomerulonefritis/patología , Glomerulonefritis Membranoproliferativa/diagnóstico , Glomerulonefritis Membranoproliferativa/enzimología , Glomerulonefritis Membranoproliferativa/patología , Glomeruloesclerosis Focal y Segmentaria/diagnóstico , Glomeruloesclerosis Focal y Segmentaria/enzimología , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Inmunohistoquímica , Riñón/patología , PronósticoRESUMEN
Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption. Emk(-/-) mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4(+)T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. As Emk(-/-) animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals.
Asunto(s)
Enfermedades Autoinmunes/enzimología , Linfocitos B/inmunología , Proteínas de Caenorhabditis elegans , Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinasas/inmunología , Linfocitos T/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Linfocitos B/citología , Linfocitos B/fisiología , Diferenciación Celular , Colon/anomalías , Femenino , Expresión Génica , Marcación de Gen , Glomerulonefritis Membranoproliferativa/enzimología , Hemoglobinuria/enzimología , Humanos , Sistema Inmunológico/inmunología , Tejido Linfoide , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Prolapso , Proteínas Serina-Treonina Quinasas/genética , Proteinuria/enzimología , Linfocitos T/citología , Linfocitos T/fisiologíaAsunto(s)
Glomerulonefritis Membranoproliferativa/enzimología , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/fisiología , Inhibidores de Proteasas/uso terapéutico , Animales , Glomerulonefritis Membranoproliferativa/tratamiento farmacológico , Glomerulonefritis Membranoproliferativa/patología , HumanosRESUMEN
Therapy of inflammation often requires the attenuation of excess cell proliferation and of extracellular matrix (ECM) accumulation. It is increasingly recognised that matrix metalloproteinases (MMP) play an important role in the regulation of these two features. For our studies, experimental mesangial proliferative glomerulonephritis and cultured mesangial cells provided the inflammation model and the opportunity to evaluate a new therapeutic strategy based on MMP inhibition. In vitro inhibition of MMP activity and synthesis successfully returned the inflammatory mesangial cell phenotype to the normally occurring resting state. In vivo, excess mesangial cell proliferation and ECM accumulation in experimental mesangial proliferative glomerulonephritis were significantly ameliorated by the use of a synthetic MMP inhibitor. Interestingly, these inhibitors lead to increased mesangial cell apoptosis. In conclusion, the antiproliferative effect of MMP inhibitors opens new perspectives in the therapy of inflammation, probably even beyond the scope of kidney diseases.
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División Celular/efectos de los fármacos , Dexametasona/farmacología , Mesangio Glomerular/efectos de los fármacos , Glomerulonefritis Membranoproliferativa/enzimología , Ácidos Hidroxámicos , Inhibidores de la Metaloproteinasa de la Matriz , Pentoxifilina/farmacología , Inhibidores de Proteasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bencilo , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Humanos , Ratas , Relación Estructura-Actividad , SuccinatosRESUMEN
Multiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN). In vitro studies demonstrate the pivotal role of mitogen-activated protein (MAP) kinases in the regulation of cellular proliferation. This study was conducted to examine whether these kinases, as a convergence point of mitogenic stimuli, are activated in mesangioproliferative GN in vivo. Therefore, anti-Thy1 GN was induced in rats using a monoclonal anti-Thy1.1 antibody (OX-7). Whole cortical tissue as well as isolated glomeruli were examined at different time points using kinase activity assays and Western blot analysis. A maximal increase in the number of glomerular mitotic figures (9.7-fold) was demonstrated 6 d after injection of the anti-Thy1.1 antibody. In parallel with this finding, a significant increase in cortical, and more dramatically glomerular, activity of extracellular signal-regulated kinase (ERK) was detected. Maximal activation of ERK was detectable on day 6. This activation of ERK was accompanied by an increase in the expression of MEK (MAP kinase/ERK kinase), the ERK-activating kinase. A marked induction of glomerular apoptosis at 2 h after injection of the anti-Thy1.1 antibody, which subsided subsequently, was demonstrated using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay as well as staining for single-stranded DNA. However, no significant activation of stress-activated protein kinase or p38 MAP kinase, both MAP kinases that are suggested to induce apoptosis and to inhibit cellular growth, was detectable at this early time point. Rather, on day 6 a dramatic decrease in the activity of p38 MAP kinase, which might have contributed to the overshooting glomerular cellular proliferation, was observed. Treatment of rats with heparin blunted glomerular proliferation as well as ERK activation and restored p38 MAP kinase activity. These observations point to ERK and p38 MAP kinase as putative mediators of the proliferative response in mesangioproliferative GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.
Asunto(s)
Mesangio Glomerular/enzimología , Glomerulonefritis Membranoproliferativa/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Activación Enzimática , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/patología , Glomerulonefritis Membranoproliferativa/inmunología , Glomerulonefritis Membranoproliferativa/patología , Heparina/farmacología , Isoanticuerpos/inmunología , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Five conditions in which the alternative pathway C3 convertase, C3b,Bb, circulates in excess as a result of factor H dysfunction are frequently accompanied by nephritis. These convertase-related nephritides are seen in association with heterozygous absence of a binding site for factor H on C3b (Marder disease), homozygous factor H deficiency, circulating factor H inhibitor, and with the nephritic factors, one of the amplification loop and the other of the terminal pathway, found in membranoproliferative glomerulonephritis (MPGN) types II and III, respectively. Observations which relate convertase to glomerular deposits are: (1) in MPGN type II, subepithelial deposits on the paramesangial segments of the glomerular basement membrane are with high frequency present in patients hypocomplementemic at biopsy, but not in those normocomplementemic; (2) in MPGN type III paramesangial deposits are similarly found with hypocomplementemia but are present for up to 1 year after normocomplementemia is achieved; (3) in MPGN type III, subendothelial deposits are present only with hypocomplementemia. The principal deposits found in factor H deficiency and in Marder disease are also paramesangial. Differences in the incidence, severity, and morphology of the nephritides accompanying convertase in excess may relate to the characteristics of the circulating convertase and/or to the C3 conversion products formed by it.
Asunto(s)
Convertasas de Complemento C3-C5/metabolismo , Glomerulonefritis Membranoproliferativa/enzimología , Factor Nefrítico del Complemento 3/fisiología , Factor H de Complemento/deficiencia , HumanosRESUMEN
Matrix metalloproteinase-2 (MMP-2, gelatinase A) is involved in the inflammatory and sclerotic events of glomerular diseases. Newly identified membrane-type matrix metalloproteinases (MT-MMP) have been shown to activate specifically proMMP-2. To date, several types of MT-MMP have been cloned; however, their expressions in glomerular diseases have not been evaluated. To investigate the role of MT-MMP in glomerular diseases, the glomerular gene expression and enzymatic activity of MT-MMP were examined during the time course of nephritis induced in rats by anti-Thy1.1 antibody injection. Both MT1-MMP and MMP-2 mRNA expression increased prominently 5 and 10 d after anti-Thy1.1 antibody injection and decreased thereafter, as assayed by semiquantitative reverse transcription-PCR. In contrast, there were no remarkable changes in the gene expression of MT2-MMP between normal and diseased tissue, and that of MT3-MMP was not detected in isolated glomeruli by reverse transcription-PCR analysis. The activation of proMMP-2 as analyzed by gelatin zymography correlated with the glomerular MT1-MMP gene expression, suggesting that proMMP-2 was activated by MT1-MMP. Protein and mRNA expression of fibronectin, one of the major mesangial matrix proteins and substrate of MMP-2, were also synchronized with MT1-MMP and MMP-2 expression. In situ hybridization revealed intense MT1-MMP mRNA expression in the proliferating mesangial cells. Interestingly, MT1-MMP gene expression exhibited a similar distribution as alpha-smooth muscle actin expression, which was closely associated with mesangial phenotypic change. These results suggest that among the newly identified MT-MMP, MT1-MMP may play the central role in activation of proMMP-2. Furthermore, the enhancement of MT1-MMP and MMP-2 expression associated with mesangial phenotypic change may contribute to the development of anti-Thy1.1 antibody-induced glomerulonephritis and remodeling of extracellular matrices.