Asunto(s)
Mesangio Glomerular/fisiología , Péptidos y Proteínas de Señalización Intracelular , Glomérulos Renales/ultraestructura , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Actinas/fisiología , Animales , Capilares/ultraestructura , Proteínas Portadoras/fisiología , Adhesión Celular , Colágeno/fisiología , Nefropatías Diabéticas/etiología , Elasticidad , Elastina/fisiología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Fibrilinas , Mesangio Glomerular/ultraestructura , Glutatión/análogos & derivados , Glutatión/biosíntesis , Humanos , Hipertensión/complicaciones , Proteínas de Unión a TGF-beta Latente , Mamíferos/anatomía & histología , Glicoproteínas de Membrana/fisiología , Ratones , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/fisiología , Permeabilidad , Ratas , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
BACKGROUND: Glomerular capillaries of the mammalian kidney are exposed to high intraluminal hydrostatic pressures and require elastic constraint to maintain size, shape, and integrity. Previous morphological and functional studies indicated that the extracellular matrices of glomeruli, that is, basement membrane and mesangial matrix, contribute to glomerular resilience and mechanical stability. Immunofluorescence microscopy findings demonstrated elastic fiber components to be located in the renal vasculature, including glomeruli. The aim of this study was to clarify the exact glomerular localization, composition, and cellular production of these proteins. METHODS: We examined the renal distribution of the elastic fiber proteins fibrillin-1, emilin, microfibril-associated glycoproteins (MAGPs) 1 and 2, latent transforming growth factor-binding protein-1 (LTBP-1), and elastin using immunohistology and immunoelectron microscopy of human, rat, and mouse kidneys. In mesangial cell cultures, we also studied the expression and extracellular deposition of such proteins by use of Northern blotting and immunocytochemistry. RESULTS: Fibrillin-1, emilin, MAGPs 1 and 2, and LTBP-1 were present in glomeruli of mouse, rat, and human kidney, where they were located predominantly in the mesangial extracellular matrix underlying glomerular endothelium and basement membrane. Several of these proteins, as well as elastin, were also expressed in the renal vasculature. While elastin localized to the glomerular vascular pole in afferent and efferent arterioles extending to Bowman's capsule, it was not found in the glomerular capillary tuft. Cultured mesangial cells of rat, mouse, and human kidneys expressed mRNAs of fibrillin-1, emilin, MAGP-2, and elastin, and the respective proteins localized within and outside of mesangial cells, as shown by immunocytochemistry. mRNA expression of fibrillin-1, emilin, and elastin was strong in quiescent mesangial cells; their gene expression was further up-regulated by transforming growth factor-beta1, while it was transiently reduced when cells were exposed to mitogenic 10% fetal calf serum and platelet-derived growth factor. CONCLUSIONS: These findings demonstrate that specific elastic fiber proteins are produced and secreted by mesangial cells. This process is regulated by growth factors. Their abundance in the extracellular matrix of the mesangium is in keeping with the concept that elastic fiber proteins contribute to the mechanical stability and elastic strength of the glomerular capillary tuft.
Asunto(s)
Proteínas Contráctiles/genética , Proteínas de la Matriz Extracelular , Mesangio Glomerular/citología , Mesangio Glomerular/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Microfilamentos/genética , Animales , Anticoagulantes/farmacología , Becaplermina , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Células Cultivadas , Proteínas Contráctiles/análisis , Elasticidad , Elastina/análisis , Elastina/genética , Células Epiteliales/química , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Matriz Extracelular/química , Matriz Extracelular/fisiología , Fibrilina-1 , Fibrilinas , Técnica del Anticuerpo Fluorescente , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Mesangio Glomerular/irrigación sanguínea , Homeostasis/fisiología , Humanos , Presión Hidrostática , Proteínas de Unión a TGF-beta Latente , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Ratones , Microcirculación/fisiología , Proteínas de Microfilamentos/análisis , Microscopía Inmunoelectrónica , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Factores de Empalme de ARN , ARN Mensajero/análisis , Ratas , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND: Extracellular matrix molecules profoundly regulate cell behavior, including proliferation. In glomerulonephritis, type I collagen accumulates in the mesangium and is constantly structurally modified and degraded during the course of the disease. METHODS: We studied how two structurally distinct forms of type I collagen, monomer versus polymerized fibrils, affect cell proliferation, mitogen-activated protein kinase (MAPK) activation, and expression of G1-phase regulatory proteins in cultured rat mesangial cells (MCs). To analyze the possible involvement of collagen-binding integrins in type I collagen-derived growth signals further, distribution patterns of integrin chains were examined by immunocytochemistry. RESULTS: Polymerized type I collagen completely prevented the increase of DNA synthesis and cell replication induced by 5% fetal calf serum (FCS) or 25 ng/mL platelet-derived growth factor (PDGF) in MCs on monomer type I collagen. Protein expression of cyclins D1 and E was markedly down-regulated in MCs plated on polymerized type I collagen for eight hours in 5% FCS, as compared with MCs on monomer type I collagen. Incubation with 5% FCS reduced expression of the cdk-inhibitor protein p27Kip1 on monomer but not on polymerized type I collagen. Moreover, polymerized type I collagen markedly reduced cyclin E-associated kinase activity in the presence of 5% FCS. Polymerized type I collagen diminished the PDGF-induced phosphorylation and nuclear translocation of p42/p44 MAPK, but did not affect phosphorylation of PDGF beta-receptors. In MCs plated on monomer type I collagen, alpha1, alpha2, and beta1 integrin chains were recruited into focal contacts. However, on polymerized type I collagen, alpha2 and beta1, but not alpha1, integrin chains were condensed into focal contacts. CONCLUSIONS: The growth-inhibitory effect of polymerized type I collagen is characterized by rapid changes of expression and/or activation of MAPK and G1-phase regulators and could result from the lack of alpha1beta1 integrin signaling in MCs on polymerized type I collagen. Conceivably, deposition of polymerized type I collagen might reflect a reparative response to control MC replication in glomerular inflammation.
Asunto(s)
Proteínas de Ciclo Celular , Colágeno/metabolismo , Fase G1/fisiología , Mesangio Glomerular/citología , Mesangio Glomerular/enzimología , Proteínas Supresoras de Tumor , Animales , Proteínas Sanguíneas/farmacología , Adhesión Celular/fisiología , Núcleo Celular/enzimología , Células Cultivadas , Colágeno/química , Ciclina E/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , ADN/biosíntesis , Matriz Extracelular/enzimología , Fase G1/efectos de los fármacos , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Hiperplasia , Integrina alfa1beta1 , Integrinas/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Polímeros/metabolismo , Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Fase S/fisiología , Transducción de Señal/fisiología , Tirosina/metabolismoRESUMEN
Regardless of the source of injury, an imbalance in the control of mesangial cell proliferation appears to play a direct role in the degree of progressive renal injury and glomerulosclerosis. Some of the regulatory mechanisms include specific soluble or non-soluble extracellular factors and a complex array of receptor-mediated signals that control the progression of the cell cycle or cell death. Understanding these regulatory processes could lead to novel therapeutic strategies to alleviate or arrest proliferative glomerular disease.
Asunto(s)
Mesangio Glomerular/patología , Glomeruloesclerosis Focal y Segmentaria/patología , Adulto , Animales , División Celular/fisiología , HumanosRESUMEN
BACKGROUND: Mesangial cell (MC) proliferation and extracellular matrix accumulation are typical responses of renal glomeruli to injury. Extracellular matrix components are known to affect MC behavior, which is mediated primarily via integrin receptors of the beta1 family. In addition to alpha1, alpha3, alpha5, and alpha6 chains of beta1 integrins, recent studies have shown the alpha8 chain to be expressed in glomeruli and renal vasculature. alpha8beta1 can serve as a receptor for fibronectin, which is abundant in the mesangium. We investigated the glomerular expression pattern of the alpha8 chain in renal tissues of mouse, rat, and humans as well as in cultured MCs. In addition, the regulation of alpha8 expression in MCs was studied in culture and in nephritic rats. METHODS: The expression of alpha8 protein in kidney tissue and cultured MCs was investigated by immunohistochemistry, immunocytochemistry, and Western blotting. The effects of TGF-beta1 on alpha8 mRNA levels in MCs were studied by Northern blot analysis. In addition, time course studies of glomerular abundance and localization of alpha8 were performed in rats with mesangioproliferative anti-Thy1.1 nephritis. RESULTS: In tissue sections of normal human, rat, and mouse kidney, we found strong immunohistochemical staining for alpha8 in the mesangium and in the media of renal arterioles. Double staining for alpha8 and Thy1.1, a surface antigen of rat MCs, showed alpha8 to be specifically expressed in MCs but not in glomerular endothelial and epithelial cells. In anti-Thy1.1 nephritis of rats, the glomerular abundance of alpha8 protein was reduced in the early mesangiolytic phase but was increased greatly with subsequent MC proliferation, peaking at day 6 of disease. At later stages of this reversible form of nephritis, the number of MCs and the extent mesangial alpha8 staining declined to control levels. Cell culture experiments revealed that freshly plated MCs organize alpha8 into focal contacts within one hour after attachment to fibronectin and vitronectin substrata, showing colocalization with focal contact proteins vinculin and talin. Stimulation of MCs with transforming growth factor-beta1 led to increases of alpha8 mRNA and protein levels. CONCLUSIONS: These results show that in human, rat, and mouse glomeruli, alpha8 integrin is strongly and exclusively expressed in MCs. Gene expression of alpha8 is regulated in cultured MCs, and alpha8 protein abundance is regulated in vivo and in MC culture. It is currently unclear what functional properties this integrin receptor protein has with regard to MC anchorage to extracellular matrix and modulation of the MC phenotype in normal and diseased glomeruli. However, in view of its abundance in the mesangium, alpha8beta1 integrin could be an important MC receptor of matrix ligands and may play a role in the embryology, physiology, and pathophysiology of the glomerular capillary tuft.
Asunto(s)
Mesangio Glomerular/fisiología , Glomerulonefritis/fisiopatología , Cadenas alfa de Integrinas , Integrinas/genética , Animales , Anticuerpos/farmacología , Northern Blotting , Western Blotting , Adhesión Celular/fisiología , División Celular/fisiología , Proteínas de la Matriz Extracelular/farmacología , Fibronectinas/farmacología , Técnica del Anticuerpo Fluorescente , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Mesangio Glomerular/química , Mesangio Glomerular/citología , Humanos , Integrinas/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Nefritis/fisiopatología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Antígenos Thy-1/inmunología , Vitronectina/farmacologíaRESUMEN
OBJECTIVE: We investigated the hypothesis that changes in blood flow in the uteroplacental and fetoplacental circulation in preeclampsia are associated with an abnormality of placental or uterine placental bed nitric oxide (NO) synthesis. METHODS: We measured pulsatility indices on Doppler waveform analysis from uterine and umbilical arteries in 20 patients with preeclampsia and 14 healthy pregnant controls before elective cesarean section. During cesarean section, biopsies from the uterine placental bed and the placenta were taken and the nitric oxide synthase (NOS) activity was measured by the [3H] L-arginine-[3H] L-citrulline conversion assay in these samples. RESULTS: The NOS activity was significantly lower in the uterine placental bed in comparison to the placental tissue (p < 0.01). Placental NOS activity was similar between patients with preeclampsia and healthy controls and in the groups with either a pathological or a normal Doppler flow in the umbilical artery. In the uterine placental bed however, NOS activity from patients with preeclampsia was significantly lower (p < 0.01), whereas the blood flow resistance in the uterine arteries was elevated (p < 0.01) in comparison to healthy controls. CONCLUSIONS: Our data show that pathological Doppler waveforms in the uterine arteries of patients with preeclampsia are paralleled by diminished NOS activity in the uterine placental bed. Therefore, the compromised NO production in the uterine placental bed may play an important role in the impaired uteroplacental blood flow and potentially in some pathological features of preeclampsia such as intervillous thrombosis formation and fetal growth retardation.
Asunto(s)
Feto/irrigación sanguínea , Óxido Nítrico Sintasa/biosíntesis , Placenta/irrigación sanguínea , Preeclampsia/fisiopatología , Ultrasonografía Prenatal , Útero/irrigación sanguínea , Adulto , Velocidad del Flujo Sanguíneo , Femenino , Feto/enzimología , Humanos , Placenta/diagnóstico por imagen , Placenta/enzimología , Embarazo , Flujo Pulsátil , Estadísticas no Paramétricas , Arterias Umbilicales/diagnóstico por imagen , Arterias Umbilicales/fisiopatología , Útero/diagnóstico por imagen , Útero/enzimologíaRESUMEN
Glomerular mesangial cells play a central role in maintaining structure and function of the glomerular capillary ultrafiltration apparatus. Under physiological and pathological conditions, mesangial cells regulate amount and composition of the surrounding extracellular matrix. Conversely, components of the embedding matrix affect the mesangial cell phenotype. These interactions are mediated via specific cell surface receptors, the best studied group of which is the beta1 integrin family. The beta1 integrins play a role in mesangial cell adhesion, migration, survival and proliferation. Expression and abundance of integrins in healthy and diseased glomeruli and their functions and mediation of signals are discussed in this review. Other factors modulating mesangial cell-matrix interactions, such as antiadhesive proteins, cytokines, disintegrins and nitric oxide, are also considered. The available evidence from in vitro and in vivo studies indicates that receptor-mediated interactions between mesangial cells and the normal or abnormal extracellular matrix regulate the mesangial cell phenotype and thus contribute to normal maintenance of the glomerulus and to remodeling and repair of the glomerular capillary tuft in response to injury.
Asunto(s)
Adhesión Celular , Mesangio Glomerular/fisiología , Glomerulonefritis/fisiopatología , Integrinas/fisiología , Enfermedades Renales/fisiopatología , Animales , Capilares/fisiología , Mesangio Glomerular/citología , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/fisiologíaRESUMEN
BACKGROUND: Mycophenolate mofetil (MMF) is used for immunosuppression after renal transplantation because it reduces lymphocyte proliferation by inhibiting inosine monophosphate dehydrogenase (IMPDH) in lymphocytes and GTP biosynthesis. In the present study we asked if therapeutic concentrations of MMF might interfere with mesangial cell (MC) proliferation which is involved in inflammatory proliferative glomerular diseases. METHODS: Rat and human MCs were growth-arrested by withdrawal of fetal calf serum (FCS) and stimulated by addition of FCS, platelet-derived growth factor (PDGF) or lysophosphatidic acid (LPA). Different concentrations of MMF (0.019-10 microM) were added concomitantly in the presence or absence of guanosine. MC proliferation was determined by [3H]thymidine incorporation. Cell viability was assessed by trypan blue exclusion. Apoptotic nuclei were stained using the Hoechst dye H33258. Cytosolic free Ca2+ concentrations were determined with the fluorescent calcium chelator fura-2-AM. RESULTS: MMF inhibited mitogen-induced rat MC proliferation with an IC50 of 0.45 +/- 0.13 microM. Human MCs proved to be even more sensitive (IC50 0.19 +/- 0.06 microM). Inhibition of MC proliferation was reversible and not accompanied by cellular necrosis or apoptosis. Addition of guanosine prevented the antiproliferative effect of MMF, indicating that inhibition of IMPDH is responsible for decreased MC proliferation. Early signalling events of GTP-binding-protein-coupled receptors, such as changes in intracellular Ca2+ levels were not affected by MMF. CONCLUSIONS: The results show that MMF has a concentration-dependent antiproliferative effect on cultured MCs in the therapeutic range, which might be a rationale for the use of this drug in the treatment of mesangial proliferative glomerulonephritis.
Asunto(s)
Mesangio Glomerular/efectos de los fármacos , Guanosina/metabolismo , Inmunosupresores/farmacología , Ácido Micofenólico/análogos & derivados , Animales , Apoptosis , Calcio/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Citosol/metabolismo , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Guanosina/farmacología , Humanos , IMP Deshidrogenasa/antagonistas & inhibidores , Arteria Ilíaca , Lisofosfolípidos/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Ácido Micofenólico/farmacología , Necrosis , Factor de Crecimiento Derivado de Plaquetas/farmacología , RatasRESUMEN
The immunosuppressant mycophenolate mofetil (MMF) inhibits the enzyme inosine-5' monophosphate dehydrogenase and thus interferes with cellular GTP synthesis. MMF suppresses the cellular and humoral immune response and has antiproliferative effects on vascular smooth muscle and mesangial cells in vitro and in vivo. In large multicenter trials with almost 1500 patients MMF has been proven highly efficacious for transplant rejection prophylaxis with the main side-effects of gastrointestinal disorders and a slightly increased incidence of viral infections. Recent investigations suggest MMF as an alternative immunosuppressant in cyclosporin A nephrotoxicity. Preliminary observations show promising results for MMF in the treatment of autoimmune-mediated renal disease. The rationale for its use in this patient group and evidence from experimental studies are discussed. As current therapy of this disease entity is still unsatisfactory, future clinical trials are necessary to investigate the efficacy and safety of MMF for this new indication.
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Inmunosupresores/uso terapéutico , Enfermedades Renales/tratamiento farmacológico , Trasplante de Riñón , Ácido Micofenólico/análogos & derivados , Ensayos Clínicos como Asunto , Humanos , Terapia de Inmunosupresión , Enfermedades Renales/inmunología , Ácido Micofenólico/uso terapéuticoRESUMEN
Glomerular mesangial cells have a central function in maintaining structure and function of the glomerular capillary ultrafiltration apparatus. Regardless of the type of glomerular injury, imbalances in the control of mesangial cell replication appear to play a key role in the pathogenesis of progressive renal failure. The available evidence from in vitro and in vivo studies indicates that such regulatory mechanisms include specific soluble and non-soluble extracellular factors and a complex array of receptor-mediated signals which control cell proliferation, survival and apoptosis. This review summarizes results from recent investigations concerning regulation of cell cycle progression in mesangial cells. In addition to results from cell culture studies, descriptive findings on expression and regulation of cell cycle-regulatory proteins and their potential role for altered mesangial cell behaviour in glomerular disease are considered. We believe that better understanding of processes which regulate mesangial cell replication could lead to novel diagnostic as well as therapeutic strategies and, thus, help control better proliferative glomerulonephritis.
Asunto(s)
Mesangio Glomerular/citología , Apoptosis , División Celular , Matriz Extracelular/fisiología , Mesangio Glomerular/fisiología , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Humanos , Insuficiencia Renal/patología , Insuficiencia Renal/fisiopatología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Mesangial cells undergo cell death both by apoptosis and necrosis during glomerular disease. Since nucleotides are released from injured and destroyed cells in the glomerulus, we examined whether extracellular ATP and its receptors may regulate cell death of cultured mesangial cells. Addition of extracellular ATP (300 microM to 5 mM) to cultured rat mesangial cells for 90 min caused a 5. 8-fold increase in DNA fragmentation (terminal deoxynucleotidyl transferase assay) and a 4.2-fold increase in protein levels of the tumor suppressor p53, which is thought to regulate apoptosis. Apoptotic DNA fragmentation was confirmed by the diphenylamine assay and by staining with the DNA-specific fluorochrome Hoechst 33258. The necrotic markers, release of lactate dehydrogenase and uptake of trypan blue, were not positive before 3 h of ATP addition. The effects of ATP on DNA fragmentation and p53 expression were reproduced by the purinergic P2Z/P2X7 receptor agonist, 3'-O-(4-benzoylbenzoyl)-ATP, and inhibited by the P2Z/P2X7 receptor blocker, oxidized ATP. Transcripts encoding the P2Z/P2X7 receptor were expressed by cultured mesangial cells as determined by Northern blot analysis. P2Z/P2X7 receptor-associated pore formation in the plasma membrane was demonstrated by the Lucifer yellow assay. We conclude that activation of P2Z/P2X7 receptors by extracellular ATP causes apoptosis and necrosis of cultured mesangial cells. Activation of purinergic P2Z/P2X7 receptors may play a role in causing death of mesangial cells during glomerular disease.
Asunto(s)
Adenosina Trifosfato/fisiología , Apoptosis/fisiología , Espacio Extracelular/metabolismo , Mesangio Glomerular/fisiología , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Mesangio Glomerular/citología , Mesangio Glomerular/efectos de los fármacos , Masculino , Necrosis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: About 20% of adults worldwide will develop hypertension. Studies and clinical observations suggest an association between hypertension and pulsatile compression of the ventrolateral medulla oblongata by a looping artery. We investigated whether neurosurgical microvascular decompression substantially decreases blood pressure long-term in patients with severe essential hypertension. METHODS: We included eight patients who had received three or more antihypertensive drugs without adequate control of blood pressure, intolerable side-effects, or both. All patients underwent microvascular decompression at the root-entry zone of cranial nerves IX and X after neurovascular compression of the ventrolateral medulla oblongata was seen on magnetic-resonance angiography. FINDINGS: 3 months after surgery, blood pressure and antihypertensive regimens had decreased substantially in three patients. Four patients who were followed up for more than 1 year became normotensive, but their antihypertensive regimens remained the same as those at 3 months. One patient did not improve. No complications associated with decompression occurred. One patient experienced a transient vocal-cord paresis after the laryngeal part of the vagus nerve was manoeuvered during surgery. INTERPRETATION: We showed a direct causal relation between raised blood pressure and irritation of cranial nerves IX and X. A subgroup of patients with essential hypertension may exist who have secondary forms of hypertension related to neurovascular compression at the ventrolateral medulla and who may be successfully treated with decompression.
Asunto(s)
Arterias Cerebrales/cirugía , Descompresión Quirúrgica/métodos , Nervio Glosofaríngeo , Hipertensión/cirugía , Bulbo Raquídeo/cirugía , Microcirugia/métodos , Síndromes de Compresión Nerviosa/cirugía , Nervio Vago , Adulto , Presión Sanguínea , Prótesis Vascular , Femenino , Humanos , Hipertensión/diagnóstico , Angiografía por Resonancia Magnética , Masculino , Bulbo Raquídeo/irrigación sanguínea , Persona de Mediana Edad , Estudios Prospectivos , Flujo Pulsátil , Resultado del TratamientoRESUMEN
Cytokine-induced nitric oxide (NO) is produced on glomerular inflammation. Glomerular injury and thrombocyte aggregation result in the release of nucleotides, which may regulate induced NO synthesis in cultured rat mesangial cells (MCs). ATP (10(-3) M) inhibited 24-h nitrite production induced by lipopolysaccharide (LPS, 10 microg/ml)/interferon-gamma (IFN-gamma, 100 U/ml) by 48.2 +/- 6. 3%, as well as induction of inducible NOS (iNOS) protein and mRNA. Also, coincubation with either 10(-4) M of UTP, ATP, or ATPgammaS inhibited LPS/IFN-gamma-induced nitrite production by 29.9 +/- 5.8, 36.4 +/- 4.3, and 50.3 +/- 6.5%, respectively, indicating involvement of purinergic P2Y2 receptors. Correspondingly, cultured MCs expressed P2Y2 receptor mRNA. Agonists for other purinergic receptors [alpha,beta-methylene-ATP, 3'-O-(4-benzoyl)-benzoyl-ATP, 2-methylthio-ATP, ADP, UDP, adenosine] were ineffective. Treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 10(-8) M) reproduced the inhibitory effect of ATP on iNOS protein expression and nitrite inhibition (by 46.6 +/- 10. 4%). The effect of ATP or PMA was reversed by the PKC inhibitors Ro-31-8220 (10(-8) M) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10(-5) M), indicating that suppression of iNOS is mediated via activation of PKC through stimulated P2Y2 receptors. In conclusion, the release of purine mediators may play a critical role for iNOS expression and synthesis of NO during glomerular inflammatory disorders.
Asunto(s)
Adenosina Trifosfato/farmacología , Mesangio Glomerular/fisiología , Interferón gamma/farmacología , Óxido Nítrico Sintasa/biosíntesis , Receptores Purinérgicos P2/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Adenosina Trifosfato/análogos & derivados , Animales , Células Cultivadas , Activación Enzimática , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/enzimología , Indoles/farmacología , Lipopolisacáridos/farmacología , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Biosíntesis de Proteínas/efectos de los fármacos , Proteína Quinasa C/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/biosíntesis , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y2 , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , omega-N-Metilarginina/farmacologíaRESUMEN
An extensive number of animal and clinical studies indicate that transforming growth factor-beta (TGF-beta s) play an important role in inflammatory and fibrotic diseases, including renal fibrosis. Recent mouse models harboring genetically engineered alterations in TGF-beta pathways reveal complicated mechanisms of regulation of TGF-beta activity in vivo. The purpose of this review is to present recent advances relevant to our understanding of the TGF-beta-signaling system in renal physiology and pathophysiology.
Asunto(s)
Enfermedades Renales/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Modelos Animales de Enfermedad , Fibrosis/metabolismo , Humanos , Ratones , Ratones Transgénicos , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
Interactions of mesangial cells (MCs) with components of the extracellular matrix (ECM) profoundly influence the MC phenotype, such as attachment, contraction, migration, survival and proliferation. Here, we investigated the effects of exogenous nitric oxide (NO) on the process of MC adhesion to ECM molecules. Incubation of rat MCs with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) dose- and time-dependently inhibited MC adhesion and spreading on various ECM substrata, being more pronounced on collagen type I than on collagen type IV, laminin or fibronectin. In contrast, SNAP did not inhibit MC adhesion to L-polylysine-coated plates. The inhibitory effects of SNAP were reduced by hemoglobin and enhanced by superoxide dismutase. The anti-adhesive action of SNAP was mimicked not only by other NO donors but also by 8-bromo-cGMP, and significantly reversed by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ). Moreover, SNAP and 8-bromo-cGMP decreased the adhesion-induced phosphorylation of focal adhesion kinase (pp125FAK). In the presence of SNAP or 8-bromo-cGMP, adherent MCs exhibited disturbed organization of alpha-actin filaments and reduced numbers of focal adhesions, as shown by immunocytochemistry. In additional experiments with adherent MCs, it was found that exposure to SNAP or 8-bromo-cGMP for 12 and 24 hours induced detachment of MCs. The results indicate that exogenous NO interferes with the establishment and maintenance of MC adhesion to ECM components. This inhibitory NO effect is mediated predominantly by cGMP-signaling. Disturbance of MC attachment to ECM molecules could represent an important mechanism by which NO affects MC behavior in vitro and in vivo.
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Mesangio Glomerular/citología , Mesangio Glomerular/efectos de los fármacos , Óxido Nítrico/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Colágeno/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Mesangio Glomerular/metabolismo , Glutatión/análogos & derivados , Glutatión/farmacología , Molsidomina/análogos & derivados , Molsidomina/farmacología , Óxido Nítrico/metabolismo , Nitroprusiato/farmacología , Compuestos Nitrosos/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , Proteínas Tirosina Quinasas/metabolismo , Ratas , S-Nitroso-N-Acetilpenicilamina , S-Nitrosoglutatión , Transducción de SeñalRESUMEN
Glomerular epithelial cells (GEC) and mesangial cells (MC) are both involved in glomerular diseases. To elucidate potential interactions between these glomerular cell types, we examined whether products of GEC affect the proliferative activity of MC. We found that cultured rat GEC secrete soluble factors into the supernate (GEC-CM) that induce proliferation of quiescent rat MC. The mitogenic activity was trypsin sensitive and partially heat-labile. Biochemical analysis of GEC-CM by gel filtration HPLC, reverse phase HPLC, and isoelectric focusing revealed at least three mitogenic fractions as well as inhibitory activity present in GEC-CM. Competitive binding assays with 125I-labeled PDGF did not show significant amounts of PDGF in GEC-CM. The biochemical features of the GEC-derived MC growth factors are distinct from IL-6, PDGF, bFGF, and endothelin, previously described GEC-derived MC growth factors. Additionally, significant contributions of known growth factors such as IL-1, IL-2, IL-3, IL-4, IL-5, TNF alpha, TGF beta, and GM-CSF are unlikely. The results indicate that GEC produce several biochemically-distinct MC growth regulators. While these epithelial cell-derived mitogens for MC require further characterization, they may play an important role in the regulation of MC replication, such as during embryogenesis and glomerular disease.
Asunto(s)
Mesangio Glomerular/citología , Sustancias de Crecimiento/fisiología , Glomérulos Renales/metabolismo , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Medios de Cultivo/farmacología , Células Epiteliales , Epitelio/metabolismo , Mesangio Glomerular/metabolismo , Glomérulos Renales/citología , Masculino , Mitógenos/farmacología , Ratas , Ratas Sprague-Dawley , Tripsina/farmacologíaRESUMEN
M. Leukemia inhibitory factor (LIF). oncostatin M (OsM) and interleukin-6 (IL-6) are members of a cytokine family, which are produced by activated macrophages and glomerular mesangial cells. These cytokines have been implicated in the pathogenesis of glomerular inflammation, but their action on glomerular cells is presently unclear. Therefore, we examined the effects of IL-6, OsM and LIF on chemokine synthesis of rat mesangial cells in culture. While LIF as well as IL-6 up-regulated monocyte chemotactic protein-1 (MCP-1) mRNA expression, OsM showed no such effect. The induction of MCP-1 mRNA by LIF and IL-6 was transient, peaking at one to two hours and two to three hours, respectively, and returning to background levels within several hours. Induction of MCP-1 mRNA by LIF and IL-6 was strongly inhibited by dexamethasone. LIF activated STAT factors in mesangial cells, suggesting their involvement in signal transduction pathways that lead to LIF-stimulated up-regulation of MCP-1 mRNA. By contrast, LIF. IL-6 and OsM failed to affect the expression of the chemokines, macrophage inflammatory protein-2 (MIP-2) and RANTES. The rapid, transient and differential regulation of MCP-1 expression induced by LIF and IL-6 contrasted with uniformly powerful effects of the proinflammatory cytokines IL-1 beta and TNF alpha that induced all tested chemokines for prolonged time periods. These results suggest that the selective and transient induction of MCP-1 by LIF and IL-6 may play a role in the preferential attraction of monocytes to the injured glomerulus.
Asunto(s)
Quimiocinas/metabolismo , Inhibidores de Crecimiento/farmacología , Interleucina-6/farmacología , Linfocinas/farmacología , Péptidos/farmacología , Animales , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CCL5/metabolismo , Quimiocina CXCL2 , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Humanos , Factor Inhibidor de Leucemia , Monocinas/metabolismo , Oncostatina M , RatasRESUMEN
BACKGROUND: In vitro studies have demonstrated that inflammatory mediators such as the cytokines TNF alpha and IL-1 upregulate or induce de novo expression of cell adhesion molecules on endothelial and epithelial cells. In the present study the expression of the cell adhesion molecules ICAM-1, VCAM-1, E-selectin and PECAM-1 was investigated in renal biopsies from patients with primary renal diseases (n = 66) and from renal allograft recipients (n = 42). METHODS: Expression of the cell adhesion molecules was determined by immunohistochemistry of frozen sections using monoclonal antibodies directed against PECAM-1, ICAM-1, VCAM-1, E-selectin and MHC class II molecules (APAAP method). RESULTS AND CONCLUSIONS: PECAM-1 and ICAM-1 were expressed in the renal vasculature and disappeared in obliterated glomeruli with endothelial cell destruction. ICAM-1 but not PECAM-1 was upregulated in renal endothelia in acute allograft rejection and inflammatory primary renal diseases. Tubular de novo expression of ICAM-1 and VCAM-1 correlated with severe structural damage of the renal parenchyma including interstitial fibrosis. Vascular and/or glomerular VCAM-1 and E-selectin expression was pronounced in severe acute allograft rejection and also reflected the intensity of inflammatory reactions in primary renal diseases with or without autoimmune disorders. De novo expression of VCAM-1 and E-selectin in renal vessels and/or glomeruli and overexpression of ICAM-1 are markers of acute and severe inflammatory processes in biopsies from allograft recipients and patients with primary renal diseases.