RESUMO
To gain insight into the glomerular capillary repair mechanisms in immunoglobulin A (IgA) nephropathy, we focused on vascular endothelial growth factor (VEGF-A) and nitric oxide (NO). Because abnormal glycosylation of serum IgA has been shown in IgA nephropathy, we examined whether VEGF-A and NO production by mesangial cells (MCs) could be modulated by aberrantly glycosylated (desialylated or degalactosylated) IgA. VEGF-A and NO synthase (NOS) gene expression were examined by reverse-transcriptase polymerase chain reaction (RT-PCR) or Northern blot analysis, and VEGF-A peptide, by capture enzyme-linked immunosorbent assay and NOS activity as production of tritium ([(3)H]) citrulline from [(3)H] arginine. Semiquantitative densitometric analysis of RT-PCR experiments showed a significant downregulation of VEGF-A messenger RNA (mRNA) in MCs incubated with aberrantly glycosylated IgA. This resulted in decreased release of VEGF-A in culture medium (P: < 0. 01). NOS activity and inducible NOS (iNOS) mRNA were enhanced by aberrantly glycosylated IgA (both P: < 0.01). No modulation of constitutive NOS mRNA was found. The depression of the VEGF-A production induced by aberrantly glycosylated IgA was mediated by NO because it was completely reversed by the NOS inhibitor, N:omega-nitro-L-arginine methyl ester. The NO donor, sodium nitroprusside, induced a bimodal modulation of VEGF; although low concentrations (0.0001 nmol/L) increased VEGF-A synthesis, greater concentrations (1,000 nmol/L) depressed it. In conclusion, we report negative control of VEGF-A synthesis in MCs by aberrantly glycosylated IgA, mediated by enhanced iNOS activity. We speculate that both increased iNOS activity and depressed VEGF-A synthesis might have a role in impairing vascular repair and favor sclerosis in IgA nephropathy.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Mesângio Glomerular/citologia , Glomerulonefrite por IGA/metabolismo , Regulação para Baixo , Fatores de Crescimento Endotelial/biossíntese , Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/metabolismo , Glomerulonefrite por IGA/diagnóstico , Humanos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio VascularRESUMO
PURPOSE: Dialytic vasculopathy is a major morbidity and mortality risk factor in patients undergoing chronic dialysis treatment. Among the pathogenetic factors some are related to the uremic condition, others are due to biocompatible reactions to dialytic materials. Endothelial cells (EC) are the target of the mediators released during bioincompatible reactions, and the related effects could be considered the initial event eliciting the vasculopathy pathogenesis. Among the others, we focused our attention on the role played in this process by the inducible isoform of nitric oxide (NO) synthase (iNOS). In previous studies we demonstrated that bioincompatible membranes, as well as acetate-containing dialysis buffers stimulate iNOS gene expression and activity in endothelial cells in culture. In this study, we planned to evaluate the potential role of a new dialysis buffer in which acetate has been substituted with HCl as a stabilizer. METHODS: ECs were incubated for 12 h at 37 degrees C with different dialysis buffers: acetate (Acet), standard bicarbonate (Bic), acetate-free buffer (AF) and HCl-bicarbonate (BicHCl). We evaluated in reverse transcriptase polymerase chain reaction (RT-PCR) the gene transcription for iNOS, the NOS activity (as the production of H3 citrulline from H3 arginine by ionic exchange chromatography), EC proliferative (H3 thymidine incorporation) and pro-apoptotic rate (TUNEL analysis) and the nuclear translocation of the transcriptional factor NF-kappaB (EMSA). RESULTS: Acetate, even in the low concentration present in Bic was able to induce a significant iNOS gene transcription (results expressed as relative units and referred to basal values: Acet 1.9 +/- 0.01 fold increase, p<0.01; Bic 1.45 +/- 0.03 p<0.05; BicHCl 1.24 +/- 0.01; AF 1.17 +/- 0.02) and translation. Acetate at concentrations both of 3 mmoL and 38 mmoL (present in the bicarbonate buffer) significantly increased the enzymatic NOS activity vs unconditioned ECs: Acet 3.46 +/- 0.3, p<0.0005; Bic 1.69 +/- 0.2, p<0.005; BicHCl 1.24 +/- 0.15; AF 1.17 +/- 0.05. The EC proliferative index was significantly depressed by acetate containing dialysis buffers (unconditioned ECs 100%, Acet 38 +/- 15%, p<0.01; Bic 65 +/- 6%, p<0.05; AF 87 +/- 8%; BicHCl 75 +/- 6%). The percentage of apoptotic ECs was significantly increased by buffers contain-ing Acet vs BicHCl and AF. Finally, acetate at the concentrations present in Acet and Bic activated and promoted the nuclear translocation of the transcriptional factor NF-kappaB in ECs (p<0.01 vs unconditioned cells). CONCLUSIONS: The acetate-free dialysis buffers have better biocompatibility and potentially down-modulate the flogistic and sclerotic processes responsible for dialytic vasculopathy.
Assuntos
Acetatos/efeitos adversos , Endotélio/citologia , Endotélio/efeitos dos fármacos , Soluções para Hemodiálise/efeitos adversos , Diálise Renal/efeitos adversos , Doenças Vasculares/etiologia , Acetatos/análise , Animais , Células Cultivadas , Soluções para Hemodiálise/química , Camundongos , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo IIAssuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/genética , Túbulos Renais/citologia , Prostaglandina-Endoperóxido Sintases/genética , Diferenciação Celular , Transformação Celular Viral , Células Cultivadas , Técnicas de Cocultura , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Fibroblastos/citologia , Humanos , Técnicas Imunoenzimáticas , Proteínas de Membrana , Monócitos/citologia , Monócitos/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Urotélio/citologiaAssuntos
Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Apoptose/efeitos dos fármacos , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Rim/citologia , Óxido Nítrico/biossíntese , Animais , Apoptose/fisiologia , Linhagem Celular , Rim/metabolismo , Camundongos , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo IIRESUMO
Since aminoglycoside efficacy is proportional to serum peak/MIC ratio and linked to post antibiotic effect, use of netilmicin once rather than twice a day has been proposed. On the other hand netilmicin might play a role in drug-induced nephrotoxicity, mainly on proximal tubule. Urinary retinol binding protein (RBP) and alpha1 microglobulin (alpha1m) are early and specific indicators of tubular damage and dysfunction. 21 preterm neonates (GA < 37 weeks) were divided in two groups on the basis of netilmicin administration modality (1: once a day, 2: twice a day, both for 7 days, at 5 mg/kg/die) and differences in netilmicin tolerability were assessed by evaluation of alpha1m and RBP levels by immunonephelometric method. No significant differences were found between the two groups either considering levels at time 1 and at time 2, or considering the difference between time 1 and 2 (Delta1/2). In our study once-daily dosing schedule shows similar low rates of nephrotoxicity, compared with multiple daily dosing schedule: this result may support the general adoption of once-daily dosing of netilmicin in clinical practice.
Assuntos
alfa-Globulinas/urina , Antibacterianos/efeitos adversos , Recém-Nascido Prematuro/urina , Túbulos Renais Proximais/efeitos dos fármacos , Netilmicina/efeitos adversos , Proteínas de Ligação ao Retinol/urina , Sepse/prevenção & controle , Antibacterianos/administração & dosagem , Esquema de Medicação , Feminino , Humanos , Recém-Nascido , Masculino , Netilmicina/administração & dosagemRESUMO
The clinical picture of acetate intolerance strictly mimics the nitric oxide (NO) effect, including smooth muscle relaxation and extreme vasodilation. Because acetate induces production of cAMP, which is a powerful stimulus of NO synthase (NOS), we evaluated the effect of different dialysate solutions with and without acetate on NOS activity in endothelial cells (EC). NOS activity of EC, evaluated as H3-citrulline produced from H3-arginine, was modulated by the dialysate composition (e.g., 38 mmol/L acetate produced an increase of 3.2 +/- 0.39-fold compared with basal values (P < 0.0005), and the small amount of acetate (4 mmol/L) in 35 mmol/L bicarbonate solution increased the NOS activity by 2 +/- 0.49-fold (P < 0.05). Conversely, the acetate-free solution produced no effect on NOS activity. The mRNA encoding for inducible NOS was highly expressed in EC incubated with acetate buffer and also with acetate in bicarbonate dialysis buffer. The EC proliferative index was depressed by acetate (P < 0.0005), and tumor necrosis factor synthesis was increased (P < 0.0005) compared with acetate-free buffer. This study suggests that dialytic "acetate intolerance" can be induced by the activation, through cAMP and tumor necrosis factor release, of NOS. The small amount of acetate in bicarbonate dialysate, although capable of inducing in vitro NOS activation, is likely to be rapidly metabolized, whereas the large amounts of this anion in acetate fluids overwhelm metabolism by the liver. Acetate-free dialysate is the only solution that provides an acceptable level of biocompatibility both in vivo and in vitro.
Assuntos
Acetatos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Óxido Nítrico/biossíntese , Animais , Soluções Tampão , Divisão Celular/efeitos dos fármacos , Soluções para Diálise/farmacologia , Tolerância a Medicamentos , Endotélio Vascular/citologia , Camundongos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/biossínteseRESUMO
BACKGROUND: The presence and the pathogenetic role of circulating IgA reacting with neutrophil cytoplasmic antigens (IgA-ANCA) in patients with Henoch-Schönlein purpura (HSP) is still debated. This study was aimed to investigate some characteristics of serum IgA and macromolecular IgA in HSP patients, focusing on IgA-ANCA. METHODS: Eighty-seven HSP patients with biopsy proved renal involvement (51 adults and 36 children) enrolled in a multicentre study of the Italian Group of Immunopathology were investigated. RESULTS: Significantly high levels of IgA immune complexes were found in both adults (P < 0.05) and children (P < 0.01), while the binding of IgA to jacalin, was significantly low in children with HSP (P < 0.01) only. Two series of ELISA were done for IgA-ANCA, in two different laboratories. Increased binding to PMN crude extracts (P < 0.01) without any modification in IgA binding to proteinase 3 was found by either specific ELISA. Conversely, the binding of IgA to myeloperoxidase (MPO) was found to be significantly (P < 0.05) increased with positive values in 25% of patients by one assay only. Three of four sera with positive IgA-MPO ANCA exhibited binding in Western-blot studies with the MPO preparation used in ELISA to a 28-kDa species. D-galactose and N-acetyl-glucosamine decreased the binding of serum IgA to MPO more in HSP than in controls (P < 0.05). CONCLUSIONS: The conflicting reports on IgA-ANCA may reflect some atypical characteristics of the reaction which can be detected only by some ELISAs. We suggest that not an antigen-antibody reaction but a lectinic interaction due to abnormal composition of IgA carbohydrate side chains may account for the IgA-ANCA reaction in patients with HSP nephritis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Vasculite por IgA/imunologia , Imunoglobulina A/metabolismo , Nefrite/imunologia , Adulto , Western Blotting , Criança , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina A/sangue , Peroxidase/metabolismoRESUMO
In 27 perinatally human immunodeficiency virus type 1 (HIV-1)-infected children, we measured, by immunonephelometry, the kappa/lambda light chain ratio (KLR) of serum immunoglobulins. The latter is a recently available laboratory index reflecting the balance between the synthesis of K isotypes and L isotypes. KLR was consistent over time in each subject, and was significantly lower than that of an age-matched normal population, independently of disease status and therapy. These data indicate a bias, in these subjects, to produce preferentially lambda rather than kappa light chains, contributing to the multiple B-cell abnormalities in HIV-1-infected children.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/biossíntese , Transmissão Vertical de Doenças Infecciosas , Síndrome da Imunodeficiência Adquirida/transmissão , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Humanos , Lactente , Masculino , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/imunologiaRESUMO
BACKGROUND: Bradykinin (BK) generation following the first contact of blood with the dialysis materials is thought to enhance hypersensitivity reactions (HSRs). Some of the effects of BK are mediated by nitric oxide (NO). We have recently reported that the pH of diluted blood modulates the kinin system. The present study was aimed to investigate the role of the pH of culture media and filter-washing solutions and BK and NO generation, either in vitro and ex vivo. METHODS: BK was measured by a specific enzyme-linked immunosorbent assay (ELISA), and NO synthase (NOS) activity by 3H-citrulline production after incubation with 3H-arginine and nitrites by using the Griess reagent. In in vitro experiments, NOS activity was detected in endothelial cells (ECs) cultured with graded BK concentrations at various pH values. Blood from 30 patients in regular dialysis was ex vivo circulated in one single passage through minifilters prerinsed with pH 7 or pH 8 phosphate buffer (PB) solutions. The out-flowing blood was tested for BK and nitrite content and was incubated with cultured ECs to evaluate its capacity to modulate NOS activity. RESULTS: BK induced in vitro a dose-dependent increase in NOS activity of ECs, which was mediated by tyrosine kinase phosphorylation. NO generation was enhanced at pH 7.2, which remained unchanged at pH 7.6. In ex vivo experiments, blood out-flowing after one passage on filters washed with pH 7 PB solutions had increased BK levels (P < 0.0001), increased nitrites (P < 0.05), and enhanced EC NOS activity (P < 0. 05) in comparison to data found when filters were washed with pH 8 PB. Only when the filters were rinsed with a solution at pH 7 did PAN DX and AN69 membranes show a distinct BK generation capability, and cuprophane a peculiar capability to enhance NOS. Such effects were prevented when dialyzers were prerinsed with pH 8 PB. Multiple regression analysis showed that the pH of the uremic blood was the driving factor for BK and NOS activation (r = 0.54, P < 0.02). CONCLUSIONS: BK and NO generation are modulated by environmental pH. Rinsing the blood and dialysate compartments of filters with an alkaline solution prior to use may mitigate the activation of mediators likely to be involved in some HSRs.
Assuntos
Álcalis/farmacologia , Bradicinina/biossíntese , Soluções para Diálise/farmacologia , Membranas Artificiais , Óxido Nítrico/biossíntese , Diálise Renal/métodos , Acidose/metabolismo , Animais , Gasometria , Bradicinina/farmacologia , Linhagem Celular , Dermatite de Contato/metabolismo , Dermatite de Contato/prevenção & controle , Endotélio/citologia , Endotélio/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Óxido Nítrico Sintase/metabolismo , Nitritos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Análise de Regressão , RoedoresRESUMO
BACKGROUND: The clinical use of cyclosporine (CsA) is limited by its nephrotoxicity. Apoptosis, perhaps instigated by increased nitric oxide synthase (NOS) activity, may play a role in such toxicity. METHODS: Human mesangial cells, human tubular cells, human umbilical vein endothelial cells, or murine endothelial cells were cultured with CsA at final concentrations of 0 to 1000 ng/mL for 4 to 24 hours. As inhibitors of apoptosis, 0.01 mol/L L-nitromethylarginine (L-NAME) or 1 microg/mL cycloheximide (CHX) was added, whereas 0.01 mol/L sodium nitroprusside (as a nitric oxide donor) was used as a positive control. Apoptosis was assessed by using TUNEL method and by DNA fragmentation by electrophoresis. In addition, NOS enzymatic activity, Northern blots for inducible NOS (iNOS) mRNA, and immunohistochemically demonstrable iNOS protein were evaluated. RESULTS: Within 12 to 24 hours, CsA significantly increased the fraction (8 to 35%) of apoptotic cells in each cell line, according to the dose. Fragmentation of DNA confirmed apoptosis. L-NAME and CHX inhibited the phenomenon, whereas sodium nitroprusside enhanced it. Each cell line significantly increased NOS activity in response to CsA, an effect blunted by L-NAME and CHX. Neither inhibitor modified the increased iNOS mRNA expression elicited by CsA. Positive staining for both iNOS and p53 proteins was observed in all cell lines incubated with CsA that were inhibited by CHX; L-NAME inhibited only p53 staining. CONCLUSIONS: CsA induces apoptosis in various renal cell lines, and this effect is mediated by the induction of iNOS via p53. These effects may contribute to the acellular fibrosis characteristic of late CsA nephrotoxicity.
Assuntos
Apoptose/fisiologia , Ciclosporina/farmacologia , Rim/efeitos dos fármacos , Rim/fisiologia , Óxido Nítrico/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Imuno-Histoquímica , Rim/citologia , Rim/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , RNA Mensageiro/metabolismoRESUMO
Nitric oxide (NO) is a powerful vasoactive product of endothelial origin, and one of its major effects is vasodilation, leading to hypotension. The role of NO in some complications of uremia is still debated. This study evaluated whether endothelial NO synthase activity could be modulated by the exposure of healthy blood to hemodialysis materials. In vitro hemodialysis sessions were performed with cuprophan and polymethylmethacrylate membranes. Blood samples from a healthy donor after recirculation for 0, 5, 15, 30, and 60 min were coincubated for 6 h with a murine endothelial cell line (t.End.1); mRNA for inducible NO synthase and enzyme activity, measured as (3H)citrulline produced from (3H)arginine, were detected. The release of interleukin (IL)-1 beta and tumor necrosis factor-alpha (TNF-alpha) from recirculating lymphomonocytes was measured, too. The NO synthase activity of endothelial cells was stimulated by blood dialyzed with cuprophan, peaking at 15 min (11-fold increase in comparison to the basal values), whereas polymethylmethacrylate was ineffective (P < 0.01 versus Cuprophan). Dialysis with cuprophan, but not with polymethylmethacrylate, induced in endothelial cells the expression of mRNA encoding for inducible NO synthase. The release of IL-1 beta and TNF-alpha after 6 h by recirculating lymphomonocytes paralleled the NO synthase activity profile in endothelial cells and was significantly higher after cuprophan exposure than after polymethylmethacrylate (P < 0.0001). In conclusion, the activity of endothelial NO synthase can be enhanced during the dialysis sessions by the interaction of lymphomonocytes with the membranes, possibly via TNF-alpha and IL-1 beta production.
Assuntos
Fenômenos Fisiológicos Sanguíneos , Membranas Artificiais , Óxido Nítrico/biossíntese , Diálise Renal/efeitos adversos , Animais , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Indução Enzimática , Interleucina-1/sangue , Camundongos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Signal transduction by mesangial cell (MC) integrins regulates cell growth and survival, extracellular matrix production, and organization. The aim of the study was to investigate human MC integrin modulation by differently glycosylated IgA and macromolecular IgA, which are thought to play a pathogenetic role in IgA nephropathy (IgAN). METHODS: MCs were incubated with purified human polymeric IgA, heat-aggregated IgA, IgA glycoforms generated by enzymatic hydrolysis of saccharide residues and serum fractions from IgAN patients, and controls isolated by lectin affinity and containing IgA with peculiar glycan patterns. Integrins were quantitated by flow cytometry. RESULTS: Cultured MCs highly expressed alphavbeta3 and some alpha3beta1; alphavbeta3 was up-regulated by matrix components (P < 0.02). In vitro desialylated and degalactosylated polymeric human IgA enhanced alphavbeta3 expression on cultured MCs (P < 0.001). Serum IgA glycoforms isolated from IgAN patients with high exposure of internal sugars, GalNAc, Neu5Ac2,6GalNAc, and Man enhanced alphav expression on cultured MCs more than healthy controls. CONCLUSIONS.: These data support the hypothesis that IgA glycation plays a role in modulating the cell-matrix interaction, and that this mechanism can be operating in IgAN.
Assuntos
Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/metabolismo , Imunoglobulina A/metabolismo , Imunoglobulina A/farmacologia , Receptores de Vitronectina/biossíntese , Anticorpos Monoclonais , Apoptose , Células Cultivadas , Matriz Extracelular/metabolismo , Citometria de Fluxo , Galactose/metabolismo , Glicosilação , Humanos , Técnicas Imunoenzimáticas , Técnicas In Vitro , Integrina alfa3beta1 , Integrinas/análise , Integrinas/biossíntese , Integrinas/imunologia , Glomérulos Renais/citologia , Glomérulos Renais/imunologia , Glomérulos Renais/metabolismo , Substâncias Macromoleculares , Ácido N-Acetilneuramínico/metabolismo , Receptores de Vitronectina/análise , Receptores de Vitronectina/imunologiaRESUMO
Hyperglycemia is considered to induce diabetic nephropathy through nonenzymatic glycation of proteins. Since hyperfiltration is likely to be the mechanism initiating the glomerular lesions, we investigated the effects of Amadori glucose adducts in serum albumin on the production of vasoactive mediators, including nitric oxide (NO) and eicosanoids, by endothelial cells (EC). Amadori adducts of glycated albumin induced a dose-response increase in NO synthase activity of murine endothelioma cells, up to 16.4 +/- 2.1-fold increase of basal values (P < 0.0001) at concentrations of 35 mg/ml mimicking physiological serum albumin concentration, and 4.6 +/- 0.8-fold increase at 17 mg/ml (P < 0.001). The effect was still detectable with glycated albumin 1.7 mg/ml, which approaches its estimated concentration in diabetic serum (1.6 +/- 0.3-fold increase, P < 0.05) The phenomenon was reproducible in human umbilical vein endothelial cells, though to a lesser extent, and further studies on murine EC were employed. The mRNA encoding for inducible NO synthase was overexpressed in EC incubated with Amadori adducts of glycated albumin in comparison to native albumin. Glycated albumin induced increased mRNA expression and synthesis of TNF-alpha. The stimulatory effect induced by glycated albumin on NO synthase activity was almost completely inhibited by anti TNF alpha antibodies. 3H-thymidine incorporation by EC was significantly inhibited when cells were grown in presence of glycated albumin (P < 0.001), and the phenomenon was abolished by the coincubation of the NO competitive inhibitor L-NAME. The early glycosylation products increased thromboxane production (P < 0.001), while prostaglandin E2 synthesis was unaffected. These data indicate that Amadori products of glycated albumin modulate NO synthase activity and eicosanoid balance in EC. These effects may be relevant to the hemodynamic changes in the early phases of diabetic nephropathy and in the lasting progression to sclerosis.
Assuntos
Produtos Finais de Glicação Avançada/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Albumina Sérica/farmacologia , Albuminas/farmacologia , Animais , DNA/biossíntese , Relação Dose-Resposta a Droga , Eicosanoides/biossíntese , Endotélio/citologia , Endotélio/efeitos dos fármacos , Endotélio/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicosilação , Humanos , Camundongos , Óxido Nítrico Sintase/biossíntese , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Veias Umbilicais/citologia , Albumina Sérica GlicadaRESUMO
BACKGROUND: This study investigated whether abnormal circulation of macromolecular IgA and IgA with altered glycosylation or electrical charge plays a role in the recurrence of IgA nephropathy (IgAN) after transplantation. STUDY DESIGN: A total of 92 renal transplant patients were enrolled; 52 IgAN patients and 40 with other non-IgAN. The IgAN group included 10 patients showing IgA mesangial deposits in the grafted kidneys (recurrent group) and 10 who did not (immunohistochemically proven non-recurrent group). In addition another 22 IgAN transplant patients were clinically free of recurrent disease. METHODS: The analyses included macromolecular IgA (IgAIC) detected by the conglutinin assay (K), heavy IgA precipitated in 2.5% polyethylene glycol (PEG), IgA-fibronectin aggregates (IgA/F Aggr), mixed IgA/IgGIC, IgA binding to mesangial matrix components (fibronectin, laminin, type IV collagen) or polycations (poly-L-lysine) and IgA with altered glycosylation (Jacalin-binding assay). RESULTS: After transplantation, IgAN patients displayed significantly higher mean levels for each variable measured than non-IgAN (ANOVA, P < 0.05). By stepwise regression analysis, the binding of IgA to fibronectin had the highest coefficient. By comparing data in recurrent and clinically non-recurrent IgAN, we observed that two groups could be distinguished by the results of the two assays for macromolecular IgA (conglutinin IgAIC and IgA-fibronectin aggregates) and IgA with increased affinity for type IV collagen (P < 0.05). When the selected group of immunohistochemically proven non-recurrent IgAN was compared to the recurrent one, a statistically significant difference was found only for the binding of IgA to type IV collagen (P < 0.05). Data from this test were significantly related with proteinuria (P < 0.05) and microscopic haematuria (P < 0.04). CONCLUSIONS: Even though the IgA serology of renal transplant IgAN patients shows peculiar features and recurrent and non-recurrent IgAN differ in many aspects, the prevalence of positive data in the two groups had no predictive value. This suggests that the recurrence of IgAN is modulated by factors affecting the interaction between circulating abnormal IgA and mesangial cells and/or matrix.
Assuntos
Glomerulonefrite por IGA/sangue , Imunoglobulina A/sangue , Análise de Variância , Sítios de Ligação , Colágeno/sangue , Feminino , Seguimentos , Glomerulonefrite por IGA/diagnóstico , Humanos , Transplante de Rim , Masculino , RecidivaRESUMO
Proteinuria represents one of the most unfavorable prognostic factors in the progression of nephropathies. Several lines of evidence support a role for proteinuria per se in the development of interstitial fibrosis, albeit the molecular mechanisms are still unknown. We investigated the potential role of integrins expressed on tubular cells in regulating the synthesis and organization of interstitial matrix or as mediators of tubulointerstitial damage in conditions mimicking the nephrotic milieu. Under basal conditions, cultured tubular cells highly expressed alpha 3 beta 1 and, at focal contacts, alpha v beta 3. In contrast, alpha v beta 5 was weakly and diffusely distributed all over the plasma membrane. Cultures on a variety of matrix substrates (fibronectin, laminin, collagen types I and IV, vitronectin, von Willebrand factor, fibrinogen) did not induce any phenotypic change in integrin expression by tubular cells. Conversely, the addition of albumin resulted in a highly increased membrane expression of beta 5, which was organized in typical focal contacts and was related to the dose of albumin added. Immunofluorescence, flow cytometry, immunoprecipitation and RT-PCR experiments argue for a complex mechanism that includes increased post-transcriptionally regulated protein synthesis, accelerated conversion of precursors to mature forms, and increased surface delivery to discrete adhesive structures. Up-regulation of the beta 5 chain in tubular cells was confirmed in 9 out of 11 kidney biopsies from proteinuric glomerulonephritides including membranous and focal sclerosing glomerulonephritis, while it was not expressed in nonproteinuric kidneys including five biopsy specimens. This is the first report indicating that proteinuria up-regulates the surface expression and distribution of a specific integrin chain on tubular cells. These observations suggest the participation of integrins in a hitherto unexplored mechanism of tubulo-interstitial responses to glomerular injury.
Assuntos
Albuminúria/metabolismo , Antígenos CD/metabolismo , Cadeias beta de Integrinas , Integrinas/metabolismo , Túbulos Renais/metabolismo , Síndrome Nefrótica/metabolismo , Proteinúria/metabolismo , Biópsia , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina G/farmacologia , Hibridização In Situ , Integrina alfaV , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fatores de TempoRESUMO
Heavy alcohol intake and/or lipotrope-deficient diet induced hepatocellular injury and mesangial deposition of IgA and often IgG in Lewis rats. The experimental animals showing more severe urinary abnormalities and histologic damage in the glomeruli had increased levels of IgA antibodies to dietary antigens and altered intestinal permeability. Based on human studies, the prolonged circulation of IgA-containing complexes associated with the liver disease could be envisaged as important for the development of mesangial IgA deposits. In order to verify this hypothesis, four groups (G) of Lewis rats were studied: G1 received thrice a weak an intragastric infusion of 1.5 ml/100 g body wt of whiskey; G2 rats were nourished with lipotrope-deficient diet; G3 rats were given both whiskey and LD diet; G4 rats were nourished with regular chow. After 12 weeks, heat-aggregated rat monomeric IgA was labeled with 133I and intravenously injected. Three control subgroups of rats, one given whiskey, one nourished with LD diet, and one with regular chow, were injected with radiolabeled heat-aggregated rat IgG. A large field-of-view digital gamma camera, equipped with an ultra-high-resolution collimator and interfaced to a dedicated computer, was used to analyze tracer kinetics and fate. The liver was the main organ involved in clearance of both test probes. The hepatic mean transit (MTT) was 11.4 +/- 11 min in G1 (proteinuria of 6.9 +/- 1.41 mg/day and hematuria +/+2), 221 +/- 19 min in G2 (proteinuria 9.1 +/- 0.64 mg/day and hematuria +2/+3), and 230 +/- 15 min in G3 (proteinuria 9.5 +/- 0.58 mg/day and hematuria +2/+3). In each case MTT value was found to be significantly prolonged compared to G4 (85 +/- 4 min). The multiple regression analysis showed that MTT values, proteinuria, and hematuria were significantly correlated (P < 0.01). Controls had trace amount proteinuria (0.82 +/- 0.17 mg/day, significantly lower than for each study group, P < 0.08) and undetectable hematuria. Similar results were obtained in control rats injected with aggregated IgG; i.e., MTT values were more prolonged in rats given whiskey or LD diet than normally nourished rats (P < 0.01). The lipotrope-deficient diet and the chronic alcohol abuse per se seem to lead to critical changes in hepatic uptake and catabolism of both an IgA and an IgG aggregate, which could account in turn for the reported appearance of renal immunoglobulin deposits in this experimental model. Due to the comparable delay in removal of IgA and IgG probes in equally nourished animals, additional factors are likely to be involved in the prominent deposition of IgA.