RESUMO
Mesangial cells stimulated with high glucose (HG) exhibit increased intracellular angiotensin II (AngII) synthesis that is correlated with the upregulation of AngII target genes, such as profibrotic cytokines. The intracrine effects of AngII can be mediated by several molecules transferred to other cells via exosomes (Exos), which play a key role in cellular communication under many physiological and pathological conditions. The aim of this study was to investigate the effects of exosomes derived from HG-stimulated human mesangial cells (HG-HMCs) on normal unstimulated HMCs. Exosomes from HMCs (C-Exos) and HG-HMCs (HG-Exos) were obtained from cell culture supernatants. HMCs were incubated with C-Exos or HG-Exos. HG stimulus induced a change in the amount but not the size of Exos. Both C-Exos and HG-Exos contained angiotensinogen and renin, but no angiotensin converting enzyme was detected. Compared with HMCs treated with C-Exos, HMCs treated with HG-Exos presented higher levels of fibronectin, angiotensinogen, renin, AT1 and AT2 receptors, indicating that HG-Exos modified the function of normal HMCs. These results suggest that the intercellular communication through Exos may have pathophysiological implications in the diabetic kidney.
Assuntos
Angiotensina II/genética , Comunicação Celular/genética , Nefropatias Diabéticas/genética , Exossomos/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Exossomos/patologia , Fibronectinas/genética , Regulação da Expressão Gênica/genética , Mesângio Glomerular/metabolismo , Glucose/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Células Mesangiais/metabolismo , Peptidil Dipeptidase A/genética , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/genética , Renina/genéticaRESUMO
BACKGROUND: Data on the risk factors for chronic kidney disease in children with immunoglobulin A nephropathy (IgAN) are scarce. This study was aimed at investigating whether glomerular C4d immunostaining is a prognostic marker in pediatric IgAN. METHODS: In this retrospective cohort study, 47 patients with IgAN biopsied from 1982 to 2010 were evaluated. Immunohistochemistry for C4d was performed in all cases. For analysis, patients were grouped according to positivity or not for C4d in the mesangial area. Primary outcome was a decline in baseline estimated glomerular filtration rate (eGFR) by 50% or more. RESULTS: Median follow-up was 8.3 years. Median renal survival was 13.7 years and the probability of a 50% decline in eGFR was 13% over 10 years. Nine children exhibited the primary outcome and 4 developed end-stage renal disease (ESRD). Compared with C4d-negative patients (n = 37), C4d-positive patients (n = 10) presented higher baseline proteinuria (1.66 ± 0.68 vs 0.47 ± 0.19 g/day/1.73 m2, p < 0.001), a progressive decline in eGFR (−10.04 ± 19.38 vs 1.70 ± 18.51 ml/min/1.73 m2/year; p = 0.045), and more frequently achieved the primary outcome (50.0 vs 10.8%, p = 0.013), and ESRD (30.0 vs 2.7%, p = 0.026). No difference was observed in Oxford classification variables. Baseline proteinuria, endocapillary hypercellularity and mesangial C4d deposition were associated with primary outcome in univariate analysis. Proteinuria and mesangial C4d deposition at baseline independently predicted the decline in eGFR. Renal survival was significantly reduced in C4d-positive patients (8.6 vs 15.1 years in C4d-negative patients, p < 0.001). CONCLUSIONS: In this exclusively pediatric cohort, positivity for C4d in the mesangial area was an independent predictor of renal function deterioration in IgAN.
Assuntos
Complemento C4b/análise , Mesângio Glomerular/patologia , Glomerulonefrite por IGA/patologia , Falência Renal Crônica/patologia , Fragmentos de Peptídeos/análise , Biomarcadores/análise , Biomarcadores/metabolismo , Biópsia , Criança , Complemento C4b/metabolismo , Progressão da Doença , Feminino , Seguimentos , Taxa de Filtração Glomerular , Mesângio Glomerular/metabolismo , Glomerulonefrite por IGA/urina , Humanos , Imuno-Histoquímica , Falência Renal Crônica/urina , Masculino , Fragmentos de Peptídeos/metabolismo , Prognóstico , Proteinúria/urina , Estudos Retrospectivos , Fatores de RiscoRESUMO
Notch pathway activation in podocytes has been shown to play an important role in diabetic kidney disease (DKD) development; however, the receptors and ligands involved in the process have not been identified. Here, we report that conditional deletion of Notch1 in podocytes using NPHS2(cre)Notch1(flox/flox) animals resulted in marked amelioration of DKD. On the contrary, podocyte-specific genetic deletion of Notch2 had no effect on albuminuria and mesangial expansion. Notch1-null podocytes were protected from apoptosis and dedifferentiation in vitro, likely explaining the protective phenotype in vivo. Deletion of Notch1 in podocytes also resulted in an increase in Notch2 expression, indicating an interaction between the receptors. At the same time, transgenic overexpression of Notch2 in podocytes did not induce phenotypic changes, while constitutive expression of Notch1 caused rapid development of albuminuria and glomerulosclerosis. In summary, our studies indicate that Notch1 plays a distinct (nonredundant) role in podocytes during DKD development.
Assuntos
Apoptose , Desdiferenciação Celular , Nefropatias Diabéticas/metabolismo , Mesângio Glomerular/metabolismo , Podócitos/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular Transformada , Células Cultivadas , Cruzamentos Genéticos , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Mesângio Glomerular/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Podócitos/patologia , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/metabolismo , Receptor Notch1/química , Receptor Notch1/genética , Receptor Notch2/química , Receptor Notch2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Prostate cancer represents the second cancer-related cause of death in North American and Chilean men. The main treatment for incurable stages of disease is surgical or pharmacological castration. However, with time and despite the addition of anti-androgens, the disease progresses to a clinical state that has been commonly referred to as hormone refractory. In recent years, the concept of hormone refractoriness has been challenged and replaced by castration resistance, acknowledging that further and optimal hormonal manipulation can be attained, beyond achieving testosterone levels at castration range. The purpose of this review is to summarize the recent therapeutic breakthroughs in the management of metastatic castrate resistant prostate cancer (mCRPC), with greater emphasis in the newer hormonal therapy agents such as Abiraterone and Enzalutamide. Future combination and sequential treatment strategies are contextualized in the current era of personalized cancer medicine and genomic characterization of prostate cancer.
Assuntos
Animais , Ratos , Angiotensina II/fisiologia , Fibronectinas/biossíntese , Células Mesangiais/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Poli(ADP-Ribose) Polimerases/fisiologia , Células Cultivadas , Fibronectinas/genética , Regulação Enzimológica da Expressão Gênica , Mesângio Glomerular/citologia , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Células Mesangiais/enzimologia , Células Mesangiais/patologia , Inibidor 1 de Ativador de Plasminogênio/genética , Poli(ADP-Ribose) Polimerases/biossíntese , Poli(ADP-Ribose) Polimerases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
INTRODUCTION: IgM nephropathy (IgMN) is a glomerulonephritis characterised by diffuse mesangial immunoglobulin M (IgM) deposits. It usually presents with nephrotic range proteinuria and, according to some previous work, it occurs most often in patients who are resistant to or dependent on steroid treatment. OBJECTIVE: To perform a clinical, histological and immunopathological description and assess the response to steroid treatment of paediatric patients diagnosed with nephrotic syndrome and diffuse mesangial IgM deposits. METHOD: This is a descriptive, retrospective study carried out in two hospitals, where the clinical records of paediatric patients with IgMN were analysed and the histological sections were re-assessed. RESULTS: thirteen children were included in this study. IgMN corresponded to 5.17% of all paediatric renal biopsies. The age of patients ranged from 1 year to 12 years (median: 2 years), 46.7% were women. The most common morphological finding was diffuse mesangial hypercellularity (46.1%), followed by focal segmental glomerulosclerosis (30.8%) and minimal glomerular changes (23.1%). All patients received steroids; in 4 cases (30.7%) as the only immunosuppressant medication, 3 (23.1%) also received cyclophosphamide, 5 (38.4%) mycophenolate, and 1 (7.7%) cyclosporine. Seven patients (53.8%) had frequent relapses, 5 (38.5%) were cortico-resistant and 1 (7.7%) cortico-dependent. Two patients (15.38%) had chronic impairment of renal function. CONCLUSION: The presence of diffuse mesangial IgM in paediatric patients with nephrotic syndrome is not a very uncommon finding; its clinical presentation has been associated with lower response to steroids. However, the long-term prognosis of these patients is still unknown.
Assuntos
Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Imunoglobulina M , Criança , Pré-Escolar , Feminino , Mesângio Glomerular/metabolismo , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/metabolismo , Humanos , Imunoglobulina M/metabolismo , Lactente , Masculino , Estudos RetrospectivosRESUMO
Previous reports have shown that angiotensin II and oxidative stress may be important features in acute poststreptococcal glomerulonephritis (APSGN) and that streptococcal erythrogenic toxin type B (ETB) and its precursor (ETBP) may have an important role in the pathogenesis of APSGN. The aim of this study was to determine the effect of ETBP on the production of angiotensin II and oxidative stress in rat mesangial cells and human mononuclear leukocytes. Mesangial cells and leukocytes were isolated from digested glomeruli and by histopaque gradient, respectively, while ETBP was isolated from nephritogenic streptococcus cultures using a cation exchange column. Angiotensin II was determined by an enzyme-linked immunosorbent assay and by cytometrics. Superoxide anion, reduced glutathione, nitrites, lipid peroxidation and catalase activity were determined by cytochemical, biochemical and enzymatic assays. Inducible nitric oxide synthase expression was determined by cytometrics. An increased production of angiotensin II was observed in ETBP-treated mesangial cell and leukocyte cultures. The ETBP induced an elevated production of superoxide anions and nitrites in mesangial cells and superoxide anions in leukocytes, while this streptococcal protein decreased the expression of inducible nitric oxide synthase in leukocytes. The ETBP was capable of inducing an increased production of angiotensin II and increased oxidative stress, both of which may be important mediators of inflammatory events in the renal tissue and during APSGN.
Assuntos
Angiotensina II/biossíntese , Proteínas de Bactérias/farmacologia , Exotoxinas/farmacologia , Mesângio Glomerular/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Animais , Catalase/metabolismo , Separação Celular , Células Cultivadas , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Glutationa/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The determinant of the diabetic nephropathy is hyperglycemia, but hypertension and other genetic factors are also involved. Glomerulus is the focus of the injury, where mesangial cell proliferation and extracellular matrix occur because of the increase of the intra- and extracellular glucose concentration and overexpression of GLUT1. Sequentially, there are increases in the flow by the poliol pathway, oxidative stress, increased intracellular production of advanced glycation end products (AGEs), activation of the PKC pathway, increase of the activity of the hexosamine pathway, and activation of TGF-beta1. High glucose concentrations also increase angiotensin II (AII) levels. Therefore, glucose and AII exert similar effects in inducing extracellular matrix formation in the mesangial cells, using similar transductional signal, which increases TGF-beta1 levels. In this review we focus in the effect of glucose and AII in the mesangial cells in causing the events related to the genesis of diabetic nephropathy. The alterations in the signal pathways discussed in this review give support to the observational studies and clinical assays, where metabolic and antihypertensive controls obtained with angiotensin-converting inhibitors have shown important and additive effect in the prevention of the beginning and progression of diabetic nephropathy. New therapeutic strategies directed to the described intracellular events may give future additional benefits.
Assuntos
Nefropatias Diabéticas/etiologia , Mesângio Glomerular , Hiperglicemia/complicações , Angiotensina II/metabolismo , Proliferação de Células/efeitos dos fármacos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Fatores Relaxantes Dependentes do Endotélio/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Mesângio Glomerular/fisiopatologia , Transportador de Glucose Tipo 1/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/fisiopatologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Esclerose/metabolismo , Esclerose/fisiopatologia , Fator de Crescimento Transformador beta1/metabolismo , Vasoconstritores/metabolismoRESUMO
O principal determinante da nefropatia diabética é a hiperglicemia, mas hipertensão e fatores genéticos também estão envolvidos. O glomérulo é o foco de lesão, onde proliferação celular mesangial e produção excessiva de matriz extracelular decorrem do aumento da glicose intracelular, por excesso de glicose extracelular e hiperexpressão de GLUT1. Seguem-se aumento do fluxo pela via dos polióis, estresse oxidativo intracelular, produção intracelular aumentada de produtos avançados da glicação não enzimática (AGEs), ativação da via da PKC, aumento da atividade da via das hexosaminas e ativação de TGF-beta1. Altas concentrações de glicose também aumentam angiotensina II (AII) nas células mesangiais por aumento intracelular da atividade da renina (ações intrácrinas, mediando efeitos proliferativos e inflamatórios diretamente). Portanto, glicose e AII exercem efeitos proliferativos celulares e de matriz extracelular nas células mesangiais, utilizando vias de transdução de sinais semelhantes, que levam a aumento de TGF-beta1. Nesse estudo são revisadas as vias que sinalizam os efeitos da glicose e AII nas células mesangiais em causar os eventos-chaves relacionados à gênese da glomerulopatia diabética. As alterações das vias de sinalização implicadas na glomerulopatia, aqui revisadas, suportam dados de estudos observacionais/ensaios clínicos, onde controle metabólico e anti-hipertensivo, especificamente com inibidores do sistema renina-angiotensina, têm-se mostrado importantes - e aditivos - na prevenção do início e progressão da nefropatia. Novas estratégias terapêuticas dirigidas aos eventos intracelulares descritos deverão futuramente promover benefício adicional.
The determinant of the diabetic nephropathy is hyperglycemia, but hypertension and other genetic factors are also involved. Glomerulus is the focus of the injury, where mesangial cell proliferation and extracellular matrix occur because of the increase of the intra- and extracellular glucose concentration and overexpression of GLUT1. Sequentially, there are increases in the flow by the poliol pathway, oxidative stress, increased intracellular production of advanced glycation end products (AGEs), activation of the PKC pathway, increase of the activity of the hexosamine pathway, and activation of TGF-beta1. High glucose concentrations also increase angiotensin II (AII) levels. Therefore, glucose and AII exert similar effects in inducing extracellular matrix formation in the mesangial cells, using similar transductional signal, which increases TGF-beta1 levels. In this review we focus in the effect of glucose and AII in the mesangial cells in causing the events related to the genesis of diabetic nephropathy. The alterations in the signal pathways discussed in this review give support to the observational studies and clinical assays, where metabolic and antihypertensive controls obtained with angiotensin-converting inhibitors have shown important and additive effect in the prevention of the beginning and progression of diabetic nephropathy. New therapeutic strategies directed to the described intracellular events may give future additional benefits.
Assuntos
Humanos , Nefropatias Diabéticas/etiologia , Mesângio Glomerular , Hiperglicemia/complicações , Angiotensina II/metabolismo , Proliferação de Células/efeitos dos fármacos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Fatores Relaxantes Dependentes do Endotélio/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Mesângio Glomerular/fisiopatologia , Transportador de Glucose Tipo 1/metabolismo , /metabolismo , Hiperglicemia/metabolismo , Hiperglicemia/fisiopatologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Esclerose/metabolismo , Esclerose/fisiopatologia , Fator de Crescimento Transformador beta1/metabolismo , Vasoconstritores/metabolismoRESUMO
Increased intrarenal renin-angiotensin system activity contributes to diabetic nephropathy. ANG II generation in mesangial cells (MC) is increased by high-glucose (HG) exposure. This study assessed the mechanisms involved in the glucose-induced ANG II generation in rat MC. Under basal conditions, MC mainly secreted prorenin. HG decreased prorenin secretion and induced a striking 30-fold increase in intracellular renin activity. After 72 h of HG exposure, only the mRNA levels for angiotensinogen and angiotensin-converting enzyme (ACE) were significantly elevated. However, after shorter periods of 24 h of HG stimulation the mRNA levels of the enzymes prorenin and cathepsin B, besides that for ACE, were significantly increased. The results suggest that the HG-induced increase in ANG II generation in MC results from an increase in intracellular renin activity mediated by at least three factors: a time-dependent stimulation of (pro)renin gene transcription, a reduction in prorenin enzyme secretion, and an increased rate of conversion of prorenin to active renin, probably mediated by cathepsin B. The increase in angiotensinogen mRNA in parallel to increased renin activity indicates that HG also increased the availability of the renin substrate. The consistent upregulation of ACE mRNA suggests that, besides renin, ACE is directly involved in the increased mesangial ANG II generation induced by HG.
Assuntos
Angiotensina II/biossíntese , Mesângio Glomerular/metabolismo , Glucose/farmacologia , Renina/metabolismo , Animais , Células Cultivadas , Meios de Cultura , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Masculino , Peptidil Dipeptidase A/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 2 de Angiotensina/biossíntese , Sistema Renina-Angiotensina/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
It was analyzed the forms of renin produced by a mouse immortalized mesangial cell line (MIC) and their ability to generate angiotensin II (AII). The synthesis, localization and secretion of renin and AII by MIC were evaluated under conditions of normal (10 mM) or high (30 mM) glucose concentration. Two major bands of 35 kDa and 70 kDa were observed in SDS-PAGE. The amino-terminal sequencing revealed the presence of prorenin and renin in these bands with higher homology with the submaxillary gland form of renin. Renin and AII were detected in cell lysate and in culture medium, indicating that MIC synthesize and secrete these peptides. Renin was localized in the cytoplasm while AII was seen predominantly inside the nucleus. High glucose induced an increase in the synthesis and secretion of renin and AII. Results suggest that MIC produce AII and a renin form similar to the submandibular. Intracellular AII may be directed at the nucleus and/or be secreted, indicating that AII may directly influences gene expression in these cells. The mechanisms of synthesis and secretion of renin and AII are potentially modified by high glucose concentration, suggesting a possible role of AII produced by mesangial cells in diabetic nephropathy.
Assuntos
Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/metabolismo , Glucose/farmacologia , Renina/metabolismo , Glândula Submandibular/química , Sequência de Aminoácidos , Angiotensina II/metabolismo , Animais , Western Blotting , Mesângio Glomerular/citologia , Imuno-Histoquímica , Manitol/metabolismo , Camundongos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Renina/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Tripsina/metabolismoRESUMO
Mesangial cells (MC) participate in the control of the glomerular function due to their ability to synthesize hormones and induce cell contraction. Since MC can produce various kinds of hormones, the purpose of the present study was to determine if they are able to synthesize catecholamines. For this evaluation, the levels of norepinephrine, epinephrine, dopamine, and biopterin, the enzymatic cofactor of tyrosine hydroxylase (TH), were analyzed by HPLC in the intracellular compartment and in the medium of primary cultured MC. To identify and locate the enzymes responsible for monoamine synthesis, TH, dopa decarboxylase, and dopamine beta-hydroxylase, Western blotting and immunocytochemistry were employed using monoclonal and polyclonal antibodies. Concentrations of NE = 57 +/- 8, EPI = 82 +/- 10, and DA = 52 +/- 9 pg/mg protein (X +/- SEM) were found in the cell homogenate. The culture medium showed concentrations of NE = 25 +/- 3, EPI = 33 +/- 3, and DA = 62 +/- 15 pg/mg protein. Western blotting analysis and immunocytochemistry evidenced the presence of all enzymes. Moreover, biopterin was also detected in the intracellular compartment and in the medium (0.28 +/- 0.03 and 5.70 +/- 2 nmol/mg cell protein, respectively). Overall, the data indicate that MC have the biosynthetic machinery necessary to produce catecholamines, suggesting that they can act as a paracrine/autocrine hormone system, contributing to the regulation of glomerular hemodynamic and renal microcirculation.
Assuntos
Catecolaminas/biossíntese , Mesângio Glomerular/metabolismo , Animais , Biopterinas/biossíntese , Células Cultivadas , Dopa Descarboxilase/metabolismo , Dopamina/biossíntese , Dopamina beta-Hidroxilase/metabolismo , Epinefrina/biossíntese , Técnicas In Vitro , Norepinefrina/biossíntese , Ratos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Bradykinin (BK) elicits extracellular-dependent [Ca2+](i) elevations in mouse mesangial cells (MMC) that are not blocked by verapamil, nifedipine, L-nicardipine, NiCl(2), or LaCl(3). The aim of the present study was to evaluate the mechanisms involved in calcium influx induced by BK in MMC. [Ca2+](i) was analyzed through spectrofluorometry employing fura-2-AM, and the data were expressed as [Ca2+](i )obtained/[Ca2+](i )basal ratio. Heparin (IP(3), a receptor antagonist) almost abolished the effects of BK in MMC (1.85 +/- 0.15 vs. 1.13 +/- 0.02, n = 4, p = 0.001). Following external and intracellular calcium store depletion, BK's effect was absent even after successful extracellular calcium replenishment. ML-7 (a myosin light chain kinase inhibitor) blocked responses to thapsigargin (2.62 +/- 0.13 vs. 1.11 +/- 0.04, n = 4, p < 0.001), but not those of BK (6.51 +/- 0.39, n = 6, vs. 5.86 +/- 1.17, n = 4, p = 0.39). On the other hand, genistein (a tyrosine kinase inhibitor) was able to inhibit thapsigargin (3.12 +/- 0.22, n = 5, vs. 1.28 +/- 0.16, n = 4, p < 0.001) as well as BK responses (6.46 +/- 0.66 vs. 2.89 +/- 0.61, n = 4, p < 0.05). Econazole (a P-450 monooxygenase inhibitor) inhibited the responses to both thapsigargin (3.45 +/- 0.16 vs. 1.03 +/- 0.03, n = 4, p < 0.001) and BK (6.49 +/- 0.83, n = 6, vs. 1.17 +/- 0.08, n = 4, p = 0.01). Finally, responses to BK were not affected by indomethacin (6.69 +/- 0.66 vs. 6.57 +/- 0.87, n = 4, p = 0.916). Thus, BK promotes an IP(3)-sensitive store-dependent calcium influx in MMC. This phenomenon seems to involve tyrosine kinase and P-450 monooxygenase products in its transduction pathway.
Assuntos
Bradicinina/farmacologia , Cálcio/metabolismo , Mesângio Glomerular/metabolismo , Animais , Anticoagulantes/farmacologia , Antifúngicos/farmacologia , Azepinas/farmacologia , Transporte Biológico/efeitos dos fármacos , Canais de Cálcio/metabolismo , Células Cultivadas , Econazol/farmacologia , Inibidores Enzimáticos/farmacologia , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Heparina/farmacologia , Camundongos , Naftalenos/farmacologia , Tapsigargina/farmacologiaRESUMO
Cathepsin B is a lysosomal thiolprotease that, because of its colocalization with renin and its ability to activate prorenin, has been proposed as a prorenin processing enzyme. To characterize the biochemical aspect of this potential cathepsin B activity in more detail, we synthesized and assayed with human cathepsin B the internally quenched fluorescent peptide Abz-FSQPMKRLTLGNTTQ-EDDnp (Abz, ortho-aminobenzoic acid fluorescent group and EDDnp, N-¿2, 4-dinitrophenyl-ethylenediamine quencher group) that contains 7 amino acids for each side of the R-L bond that is the processing site of human prorenin. Human cathepsin B hydrolyzed this peptide at the correct site (R-L bond), with k(cat)/K(m)=75 mmol/L(-1) s(-1). Analogues of this peptide obtained by Ala scanning at positions P(5) to P(5)' were also synthesized and assayed as substrates for human cathepsin B. The obtained specificity constant (k(cat)/K(m)) values have a significant parallel with the previous data of prorenin activation by AtT-20 cells and in vitro by cathepsin B. In addition, we demonstrated the presence of cathepsin B-like activity in rat mesangial cells and the ability of its whole soluble fraction lysates, as well as that of purified cloned rat cathepsin B, to hydrolyze Abz-IKKSSF-EDDnp at the K-S bond, which contains 6 amino acids of rat prorenin processing site. The specificity data of cathepsin B toward peptides derived from prorenin processing site support the view that human or rodent cathepsin B could be involved in the intracellular processing of prorenin that is locally synthesized or taken up from the extracellular compartment.
Assuntos
Catepsina B/metabolismo , Precursores Enzimáticos/metabolismo , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Renina/metabolismo , Animais , Células Cultivadas , Precursores Enzimáticos/química , Fluorescência , Mesângio Glomerular/citologia , Mesângio Glomerular/metabolismo , Humanos , Hidrólise , Processamento de Proteína Pós-Traducional , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Renina/químicaRESUMO
Bradykinin (BK) induces increases in cytosolic calcium concentration [Ca++]i in several cell lines. Because the role of BK in the renal system, particularly in mesangial cell (MC), is not clear, we investigated the effects of kinins on [Ca++]i in mouse-immortalized MC. [Ca++]i was evaluated by spectrofluorometry and expressed as a ratio between the obtained and basal [Ca++]i. BK (0.1 microM) induced a non-sustained increase in [Ca++]i (4.70 +/- 0.27; N = 28). A similar effect was observed with the B2 receptor agonist, Tyr8-BK (0.1 microM, 3.34 +/- 0.48; N = 7), while B1 receptor agonists, des-Arg10-Kallidin (Kal) (1 microM, N = 11) and des-Arg9-BK (1 microM, N = 8), exhibited only discrete responses (1.45 +/- 0.08 and 1.12 +/- 0.04, respectively). Cross-desensitization was seen between BK and Tyr8-BK, but not between BK and des-Arg10-Kal. The BK response was decreased (5.09 +/- 0.30, N = 6 to 1.57 +/- 0.12, N = 7, P < 0.001) by the B2 receptor antagonist HOE 140 (0.1 microM, 15 min), while the B1 receptor antagonist des-Arg9-[Leu8]-BK (1 microM, 15 min) had no effect on BK or des-Arg10-Kal actions. Incubation of cells with Escherichia coli lipopolysaccharide (100 microg/ml, 24 h) alone or in association with tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml, N = 6) did not enhance B1 agonist responses. BK was inhibited by repeated cell washouts in zero Ca++ solution (2.04 +/- 0.19, N = 6 P < 0.001), and the residual response was almost abolished by thapsigargin (Thaps) a sarcoplasmic reticulum (SR) calcium-ATPase inhibitor (1 microM) (1.18 +/- 0.08, N = 5 P < 0.001). Additionally, BK was not inhibited by verapamil (50 microM), nifedipine (30 microM), Ni++ (300 microM) or La (10 microM). In conclusion, BK induces [Ca++]i in mouse MC mainly by B2 receptor activation. B1 receptors have a minor role in this phenomenon even in the presence of known B1 receptor synthesis inducers. Finally, BK mobilizes extracellular calcium sources and, to a lesser extent, intracellular Thaps-sensitive calcium stores. The ion channels involved in calcium influx remain to be detected.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/metabolismo , Cininas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Transformada , Citosol/efeitos dos fármacos , Mesângio Glomerular/citologia , Camundongos , Receptores da Bradicinina/metabolismoRESUMO
The structural organization of the renal corpuscle (RC), ciliated neck segment (NS) and the proximal tubule (PT) were studied in the toad, Bufo arenarum, by means of light and transmission electron microscopy. The ciliated neck segment and the proximal tubule are located in the dorsolateral zone of the kidney, while the distal tubules are located in a ventromedial zone. RC are found between these two zones. The glomerular filter apparatus consists of the podocyte epithelium, a basement membrane, a subendothelial space and an endothelium. The podocyte emits cytoplasmatic processes extending over the surface of the glomerular capillaries. These processes divide into further processes ending in expansions known as pediceles. The basement membrane consists of a lamina rara externa and a rather thin lamina densa, while the subendothelial space contains collagen fibers and slender cytoplasmic processes of the mesangial cells. NS are composed of ciliated cells with a characteristic location of the mitochondria. The PT consists of prismatic cells with a dense luminal brush border of long microvilli and numerous apical vesicles. The basal cell membrane is increased by small infoldings. One characteristic structure of the cytoplasm is the presence of lipid droplets. The cytological structure of PT cells can be considered as an adaptation for the reabsorption of organic materials
Assuntos
Animais , Masculino , Feminino , Bufonidae/anatomia & histologia , Bufonidae/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/ultraestrutura , Mesângio Glomerular/ultraestrutura , Mesângio Glomerular/metabolismoRESUMO
The structural organization of the renal corpuscle (RC), ciliated neck segment (NS) and the proximal tubule (PT) were studied in the toad, Bufo arenarum, by means of light and transmission electron microscopy. The ciliated neck segment and the proximal tubule are located in the dorsolateral zone of the kidney, while the distal tubules are located in a ventromedial zone. RC are found between these two zones. The glomerular filter apparatus consists of the podocyte epithelium, a basement membrane, a subendothelial space and an endothelium. The podocyte emits cytoplasmatic processes extending over the surface of the glomerular capillaries. These processes divide into further processes ending in expansions known as pediceles. The basement membrane consists of a lamina rara externa and a rather thin lamina densa, while the subendothelial space contains collagen fibers and slender cytoplasmic processes of the mesangial cells. NS are composed of ciliated cells with a characteristic location of the mitochondria. The PT consists of prismatic cells with a dense luminal brush border of long microvilli and numerous apical vesicles. The basal cell membrane is increased by small infoldings. One characteristic structure of the cytoplasm is the presence of lipid droplets. The cytological structure of PT cells can be considered as an adaptation for the reabsorption of organic materials.
Assuntos
Bufonidae/anatomia & histologia , Glomérulos Renais/ultraestrutura , Túbulos Renais Proximais/ultraestrutura , Animais , Bufonidae/metabolismo , Feminino , Mesângio Glomerular/metabolismo , Mesângio Glomerular/ultraestrutura , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , MasculinoRESUMO
To evaluate functional alterations of mesangial cells induced by diabetes (DMC), we observed the changes of cytosolic calcium ([Ca]i) in response to the vasoconstrictor agonists angiotensin II (Ang II) and norepinephrine (NOR). DMC were obtained from rats with streptozotocin-induced diabetes, cultured in normal medium and identified as mesangial cells (MC) in the third subculture. [Ca]i was measured using fura-2 as a fluorophore. Basal calcium levels (60 to 80 nM) in DMC were not different from control mesangial cells (CMC). The high glucose (30 mM) medium concentration reduced the response of CMC and DMC to Ang II and NOR. This was not an osmotic effect since mannitol did not alter these responses. When DMC were stimulated with Ang II, a desensitized response was always observed, with a transient variation of [Ca]i (N = 6, P < 0.05). In contrast, a non-desensitized response with a sustained pattern of [Ca]i increases was obtained in NOR-stimulated DMC. Therefore, the present results suggest that DMC show a modified response to stimulation of the Ang II receptor, which is expressed phenotypically in culture by desensitization. Furthermore, these alterations induced by diabetes environment in MC in vivo were maintained in vitro despite a long period (approximately 5 months) in which the cells were grown in normal culture medium.
Assuntos
Angiotensina II/farmacologia , Cálcio/metabolismo , Mesângio Glomerular/citologia , Norepinefrina/farmacologia , Vasoconstritores/farmacologia , Animais , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Citosol/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Relação Dose-Resposta a Droga , Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/metabolismo , Glucose/farmacologia , Masculino , Ratos , Ratos WistarRESUMO
In the renal glomerulus, two extracellular matrices have been identified, the glomerular basement membrane and the mesangial matrix. Accumulation of glomerular extracellular matrix is a conspicuous feature of most forms of progressive glomerular disease, including diabetic nephropathy. Since proteoglycans are prominent components of the extracellular matrix, we examined the glycosaminoglycans and proteoglycans synthesized in vitro by mesangial cells from normal and diabetic rats. A mixture of dermatan sulfate and heparan sulfate was recovered. Dermatan sulfate was the predominant glycosaminoglycan synthesized and most of it was released to the culture medium, in contrast to heparan sulfate which was found to be cell associated to a higher degree. The dermatan sulfate chains are composed by D-glucuronic and L-iduronic acid-containing disaccharides and are highly sulfated. Mesangial cells from diabetic rats produce much more glycosaminoglycans than mesangial cells from normal rats, especially dermatan sulfate and this increase was proportional to the duration of diabetes. In contrast, exposure of mesangial cell from normal rats to elevated glucose did not lead to any changes in glycosaminoglycan synthesis, indicating that this short-term culture conditions may not adequately simulate diabetes mellitus. Other factors related to diabetes environment may be responsible for the observed alterations. The dermatan sulfate was secreted to the medium as proteoglycan. Two dermatan sulfate proteoglycans were identified, with molecular weights of 120 and 85 kDa respectively. The proteoglycan core protein M(r) was 45 kDa and the dermatan sulfate chains were 35 kDa. It is possible that the two proteoglycans represent two populations, one with two dermatan sulfate side chains (120 kDa) and the other with only one side chain (85 kDa), presumably fitting in the decorin/biglycan family of small proteoglycans.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Mesângio Glomerular/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/patologia , Glucose/farmacologia , Glicosaminoglicanos/biossíntese , Masculino , Proteoglicanas/biossíntese , Ratos , Ratos Wistar , Radioisótopos de EnxofreRESUMO
BACKGROUND: The participation of monocytes-macrophages and their products in the pathogenesis of several types of glomerulonephritis has become increasingly evident. One of the most important aspects is the potential stimulation of monocyte-macrophages of the extracellular matrix. Therefore, production of fibronectin (FN) by glomeruli of rats with nephrotoxic nephritis was studied. In addition, the effect of macrophage-conditioned medium and interleukin-1 on FN production by cultured mesangial cell (MC) was tested. EXPERIMENTAL DESIGN: Nephrotoxic nephritis was induced in rats by injection of nephrotoxic serum and glomeruli obtained at different periods of time from nephritic and normal animals were cultured. FN in glomerular supernatants was determined by enzyme-linked immunosorbent assay and newly synthesized FN by incorporation of [35S]-methionine. Macrophage-conditioned medium was obtained from cultures of peritoneal resident macrophages and elicited macrophages by different stimuli. MC were cultured with or without macrophage supernatant or interleukin-1 and FN content from MC supernatants was determined by enzyme-linked immunosorbent assay. RESULTS: These data show increase amounts of FN in nephritic cultures when compared with saline controls (time of nephritis; day 4: 3.9-, day 8: 4.35-, and day 18: 2.68-fold increase in FN) and experiments of newly synthesized FN by incorporation of [35S]-methionine had similar results. Macrophage-conditioned medium had FN stimulatory effect on cultured MC but interleukin-1 did not. CONCLUSIONS: These data suggest: (a) that there is an increased newly synthesized FN production in glomeruli from rats with nephrotoxic nephritis, (b) that macrophage produce a FN-stimulatory factor(s) for MC, and (c) that this stimulatory factor probably is not interleukin-1.
Assuntos
Fibronectinas/biossíntese , Mesângio Glomerular/metabolismo , Glomérulos Renais/metabolismo , Macrófagos/fisiologia , Nefrite/metabolismo , Animais , Feminino , Imunofluorescência , Mesângio Glomerular/patologia , Soros Imunes/intoxicação , Interleucina-1/farmacologia , Rim/efeitos dos fármacos , Glomérulos Renais/patologia , Nefrite/patologia , Ratos , Ratos Endogâmicos Lew , Proteínas RecombinantesRESUMO
1. The distribution and amount of ferritin in the glomeruli following intravenous injection of radiolabeled ferritin (125I-ferritin) was studied in 25 normal rats and in 25 rats with membranous nephropathy. The animals used were male Sprague-Dawley rats weighing 180-200 g at the beginning of the experiment. Membranous nephropathy was induced by repeated iv injections of 1.0 mg cationic bovine serum albumin during 28 days. 2. At the end of the experiment the animals received 125I-ferritin iv and were sacrificed 2, 6, 12, 24 and 36 h later, and the glomeruli were isolated. 3. Mean (+/- SEM) levels of 125I-ferritin in the glomeruli reported as cpm/mg protein in rats injected with cationic bovine serum albumin were: 731.8 +/- 155.6 after 2 h, 946.4 +/- 268.2 after 6 h, 565.4 +/- 143.5 after 12 h, 251.8 +/- 26.5 after 24 h, and 202 +/- 29.1 after 36 h. Mean (+/- SEM) 125I-ferritin in normal rats were: 2 h: 256.2 +/- 44.6; 6 h: 214.2 +/- 8.78; 12 h: 198.2 +/- 32.2; 24 h: 51.5 +/- 3.57; 36 h: 40.6 +/- 5.48. 125I-ferritin levels in the glomeruli isolated from rats injected with cationic bovine serum albumin were significantly higher than in control rats at 2, 6, 24 and 36 h. 4. The distribution of ferritin in the glomeruli was studied by a direct immunofluorescence technique. Normal and nephrotic rats showed ferritin in the glomerular mesangium only, with similar pattern and intensity. 5. These data show that rats with membranous glomerulonephritis induced by cationic bovine serum albumin presented an increased macromolecule uptake by the glomerular mesangium. However, the mechanism underlying this mesangial overloading is still unknown.