RESUMEN

AIMS/HYPOTHESIS: Finding ways to stimulate the regeneration of endogenous pancreatic beta cells is an important goal in the treatment of diabetes. Parathyroid hormone-related protein (PTHrP), the full-length (1-139) and amino-terminal (1-36) peptides, enhance beta cell function, proliferation, and survival. Therefore, we hypothesize that PTHrP(1-36) has the potential to regenerate endogenous beta cells. METHODS: The partial pancreatectomy (PPx) mouse model of beta cell injury was used to test this hypothesis. Male Balb/c mice underwent either sham-operation or PPx, and were subsequently injected with PTHrP(1-36) (160µg/kg) or vehicle (veh), for 7, 30, or 90 days. The four groups of mice, sham-veh, sham-PTHrP, PPx-veh, and PPx-PTHrP were assessed for PTHrP and receptor expression, and glucose and beta cell homeostasis. RESULTS: PTHrP-receptor, but not the ligand, was significantly up-regulated in islets from mice that underwent PPx compared to sham-operated mice. This suggests that exogenous PTHrP could further enhance beta cell regeneration after PPx. PTHrP did not significantly affect body weight, blood glucose, plasma insulin, or insulin sensitivity, in either sham or PPx mice. Glucose tolerance improved in the PPx-PTHrP versus PPx-veh mice only in the early stages of treatment. As hypothesized, there was a significant increase in beta cell proliferation in PPx-PTHrP mice at days 7 and 30; however, this was normalized by day 90, compared to PPx-veh mice. Enhanced beta cell proliferation translated to a marked increase in beta cell mass at day 90, in PPx-PTHrP versus PPx-veh mice. CONCLUSIONS: PTHrP(1-36) significantly enhances beta cell regeneration through increased beta cell proliferation and beta cell mass after PPx. Future studies will determine the potential of PTHrP to enhance functional beta cell mass in the setting of diabetes.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Pancreatectomía , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Insulina/metabolismo , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos BALB C , Receptor de Hormona Paratiroídea Tipo 1/biosíntesis , Regeneración , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Sickle cell anemia (SCA) is an inherited red blood cells (RBC) disorder characterized by significantly decreased RBC deformability. The present study aimed to assess whether modulation of RBC Nitric Oxide Synthase (RBC-NOS) activation could affect RBC deformability in SCA.Blood of twenty-five SCA patients was treated for 1 hour at 37°C with Phosphate Buffered Saline (PBS) or PBS containing 1% of Dimethylsulfoxyde as control, L-arginine or N(5)-(1-Iminoethyl)-L-ornithine (L-NIO) to directly stimulate or inhibit RBC-NOS, insulin or wortmannin to indirectly stimulate or inhibit RBC-NOS through their effects on the PI3 Kinase/Akt pathway, and sodium nitroprusside (SNP) and 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as NO donor and NO scavenger, respectively. RBC deformability was measured by ektacytometry at 3 Pa.RBC deformability significantly increased after insulin treatment and significantly decreased after L-NIO and wortmannin incubation. The other conditions did not affect deformability. Significantly increased nitrotyrosine levels, a marker of enhanced free radical generation, were detected by immunohistochemistry in SNP and insulin treated samples.These data suggest that RBC deformability of SCA can be modulated by RBC-NOS activity but also that oxidative stress may impair effectiveness of RBC-NOS produced NO.

Asunto(s)

Anemia de Células Falciformes/sangre , Deformación Eritrocítica/fisiología , Eritrocitos/metabolismo , Óxido Nítrico/metabolismo , Anemia de Células Falciformes/metabolismo , Eritrocitos/citología , Femenino , Humanos , Inmunohistoquímica , Masculino , Estrés Oxidativo/efectos de los fármacos

RESUMEN

Vaso-occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in-vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy.

Asunto(s)

Anemia de Células Falciformes/sangre , Viscosidad Sanguínea/efectos de los fármacos , Agregación Eritrocitaria/efectos de los fármacos , Membrana Eritrocítica/metabolismo , Poloxámero/farmacología , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/patología , Adhesión Celular/efectos de los fármacos , Membrana Eritrocítica/patología , Femenino , Humanos , Masculino

RESUMEN

The aim of the present study was to test the effects of Pfaffia paniculata (PP) extract on the red blood cell (RBC) rheological properties of patients with sickle cell disease (SCD) and healthy (AA) individuals. Blood from 7 SCD and 4 AA individuals were collected in EDTA tubes. Washed RBCs were incubated with various concentration of PP extract: 0.0, 0.2 or 0.5 mg/ml of PP solution for 5 hrs at 37°C. RBC deformability was measured by ektacytometry at 9 shear stresses ranging from 0.3 to 30 Pa, and RBC aggregation properties were determined by laser-backscattered techniques. Because RBCs from SCD patients are fragile, a stability test was also performed to test for the fragility of RBC exposed to a constant shear stress (70 Pa) for 10 min. While RBC deformability was not improved by the use of PP extract in AA, we noted an improvement of this parameter in patients with SCD between the 0.0 and 0.5 mg/ml conditions. In contrast to AA RBCs, the fragility of SCD RBCs was not affected by PP extract. In conclusion, this study demonstrates the beneficial effects, in-vitro, of PP extract on the RBC deformability of SCD patients, notably at high shear stress (a shear stress condition usually found in capillaries).

Asunto(s)

Anemia de Células Falciformes/sangre , Deformación Eritrocítica/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Extractos Vegetales/uso terapéutico , Reología , Femenino , Humanos , Masculino

RESUMEN

Human red blood cells (RBC) express an active and functional endothelial-like nitric oxide (NO) synthase (RBC-NOS). We report studies on RBC-NOS activity in sickle cell anaemia (SCA), a genetic disease characterized by decreased RBC deformability and vascular dysfunction. Total RBC-NOS content was not significantly different in SCA patients compared to healthy controls; however, using phosphorylated RBC-NOS-Ser(1177) as a marker, RBC-NOS activation was higher in SCA patients as a consequence of the greater activation of Akt (phosphorylated Akt-Ser(473) ). The higher RBC-NOS activation in SCA led to higher levels of S-nitrosylated α- and ß-spectrins, and greater RBC nitrite and nitrotyrosine levels compared to healthy controls. Plasma nitrite content was not different between the two groups. Laser Doppler flowmetric experiments demonstrated blunted microcirculatory NO-dependent response under hyperthermia in SCA patients. RBC deformability, measured by ektacytometry, was reduced in SCA in contrast to healthy individuals, and pre-shearing RBC in vitro did not improve deformability despite an increase of RBC-NOS activation. RBC-NOS activation is high in freshly drawn blood from SCA patients, resulting in high amounts of NO produced by RBC. However, this does not result in improved RBC deformability and vascular function: higher RBC-NO is not sufficient to counterbalance the enhanced oxidative stress in SCA.

Asunto(s)

Anemia de Células Falciformes/enzimología , Deformación Eritrocítica , Eritrocitos/enzimología , Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo , Anemia de Células Falciformes/patología , Activación Enzimática , Eritrocitos/patología , Femenino , Humanos , Masculino , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Espectrina/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo

Asunto(s)

Eritrocitos/enzimología , Enfermedad de la Hemoglobina SC/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Estudios de Casos y Controles , Activación Enzimática , Deformación Eritrocítica , Eritrocitos/patología , Regulación de la Expresión Génica , Enfermedad de la Hemoglobina SC/genética , Enfermedad de la Hemoglobina SC/patología , Humanos , Óxido Nítrico Sintasa de Tipo III/genética , Transducción de Señal , Espectrina/metabolismo

RESUMEN

INTRODUCTION: Experimental studies have suggested that indoxyl sulfate (IS), a protein-bound uremic toxin, may be involved in the development of renal osteodystrophy. OBJECTIVE: evaluate the association between IS levels and biochemical parameters related to mineral metabolism and bone histomorphometry in a cohort of pre-dialysis chronic kidney disease (CKD) patients. METHODS: This is a post-hoc analysis of an observational study evaluating the association between coronary calcification and bone biopsy findings in 49 patients (age: 52 ± 10 years; 67% male; estimated glomerular filtration rate: 36 ± 17 ml/min). Serum levels of IS were measured. RESULTS: Patients at CKD stages 2 and 3 presented remarkably low bone formation rate. Patients at CKD stages 4 and 5 presented significantly higher osteoid volume, osteoblast and osteoclast surface, bone fibrosis volume and bone formation rate and a lower mineralization lag time than CKD stage 2 and 3 patients. We observed a positive association between IS levels on one hand and the bone formation rate, osteoid volume, osteoblast surface and bone fibrosis volume on the other. Multivariate regression models confirmed that the associations between IS levels and osteoblast surface and bone fibrosis volume were both independent of demographic and biochemical characteristics of the study population. A similar trend was observed for the bone formation rate. CONCLUSION: Our findings demonstrated that IS is positively associated with bone formation rate in pre-dialysis CKD patients.

Asunto(s)

Huesos/anatomía & histología , Indicán/sangre , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Diálisis Renal , Insuficiencia Renal Crónica/sangre

RESUMEN

Introduction: Experimental studies have suggested that indoxyl sulfate (IS), a protein-bound uremic toxin, may be involved in the development of renal osteodystrophy. Objective: evaluate the association between IS levels and biochemical parameters related to mineral metabolism and bone histomorphometry in a cohort of pre-dialysis chronic kidney disease (CKD) patients. Methods: This is a post-hoc analysis of an observational study evaluating the association between coronary calcification and bone biopsy findings in 49 patients (age: 52 ± 10 years; 67% male; estimated glomerular filtration rate: 36 ± 17 ml/min). Serum levels of IS were measured. Results: Patients at CKD stages 2 and 3 presented remarkably low bone formation rate. Patients at CKD stages 4 and 5 presented significantly higher osteoid volume, osteoblast and osteoclast surface, bone fibrosis volume and bone formation rate and a lower mineralization lag time than CKD stage 2 and 3 patients. We observed a positive association between IS levels on one hand and the bone formation rate, osteoid volume, osteoblast surface and bone fibrosis volume on the other. Multivariate regression models confirmed that the associations between IS levels and osteoblast surface and bone fibrosis volume were both independent of demographic and biochemical characteristics of the study population. A similar trend was observed for the bone formation rate. Conclusion: Our findings demonstrated that IS is positively associated with bone formation rate in pre-dialysis CKD patients. .

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Introdução: Estudos experimentais indicam que o indoxil sulfato (IS), uma toxina urêmica ligada à proteína, pode estar envolvido no desenvolvimento da osteodistrofia renal. Objetivo: Avaliar a associação entre os níveis séricos de IS e parâmetros bioquímicos do metabolismo mineral e da histomorfometria óssea em uma coorte de pacientes com doença renal crônica (DRC) pré-diálise. Métodos: Análise post-hoc de um estudo que avaliou a associação entre calcificação coronariana e histomorfometria óssea em 49 pacientes (idade: 52 ± 10 anos; 67% sexo masculino; taxa de filtração glomerular estimada: 36 ± 17 ml/min). Os níveis séricos de IS foram dosados. Resultados: Pacientes com DRC estágio 2 e 3 apresentaram uma taxa de formação óssea baixa. Pacientes com DRC estágio 4 e 5 apresentaram volume osteoide, superfícies osteoblástica e osteoclástica, volume de fibrose e taxa de formação óssea significativamente maiores e intervalo de mineralização significativamente menor que os pacientes com DRC estágio 2 e 3. Os níveis séricos de IS associaram-se positivamente com a taxa de formação óssea, volume osteoide, superfície osteoblástica e volume de fibrose. A análise de regressão multivariada identificou que o IS é um fator independente determinante da superfície osteoblástica e fibrose. Uma tendência similar foi observada para a taxa de formação óssea. Conclusão: Nosso estudo sugere que, na DRC pré-dialítica, o IS correlaciona-se positivamente com a formação óssea. .

Asunto(s)

Femenino , Humanos , Masculino , Persona de Mediana Edad , Huesos/anatomía & histología , Indicán/sangre , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Estudios Transversales , Diálisis Renal , Insuficiencia Renal Crónica/sangre

RESUMEN

BACKGROUND: Patients with chronic kidney disease (CKD) develop various bone abnormalities characterized by impaired bone remodelling. Recent data suggest that accumulation of the uraemic toxin indoxyl sulphate (IS) may be one of the factors involved in bone abnormalities in CKD patients. Indeed, it was recently reported that IS induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells. However, it is not yet known whether IS also affects osteoclast cells. METHODS: In the present study, we assessed the direct effect of IS at uraemic concentrations and in the presence (to reach the 3 mM concentration) or absence of added inorganic phosphate (Pi) on osteoclast (OCL) differentiation and bone-resorbing activity in two well-established cellular models of monocyte/macrophage (peripheral blood mononuclear cells and the RAW 264.7 cell line). RESULTS: We found that IS inhibits both OCL differentiation and bone-resorbing activity in a dose-dependent manner and that these effects were enhanced in the presence of Pi at 3mM concentration. IS induced a gradual inhibition of JNK, Akt, p38, ERK1/2 phosphorylation and AP-1 DNA-binding activity. The effects of IS on OCL differentiation and AP-1 were prevented by probenecid, a competitive inhibitor of organic anion transporters, suggesting that IS's effects occur subsequently to its intake. CONCLUSION: Our findings strongly suggest that IS not only inhibits osteoblast function but also has an inhibitory effect on OCL function and thus could affect bone remodelling in CKD patients.

Asunto(s)

Resorción Ósea/tratamiento farmacológico , Diferenciación Celular/efectos de los fármacos , Indicán/farmacología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Animales , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Osteoclastos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Uremic syndrome is attributed to the progressive retention of a large number of compounds, such as indoxyl sulfate, which under physiological conditions are excreted by the kidneys. Previous in vitro studies have demonstrated that uremic indoxyl sulfate concentrations induce a weak increase in the proliferation of both rat and human vascular aortic smooth muscle cells (hVASMC) after short term exposition to the toxin (i.e. 24 h). In the present study, we evaluated indoxyl sulfate effects on the proliferation of hVASMC at three different concentrations after long-term exposure (seven days). In contrast to previously published studies, we observed a dose-dependent and significant inhibitory effect of this toxin on hVASMC proliferation. We also demonstrated that indoxyl sulfate inhibits epidermal growth factor-induced hVASMC proliferation after long-term exposure. Indoxyl sulfate effects were associated with a dose-dependent induction of intracellular reactive oxygen species and up-regulation of p21 and p27 protein expression. Chronic exposure to indoxyl sulfate produces a significant inhibitory effect on hVASMC proliferation. The relevance of these findings must be evaluated by further studies, particularly in an in vivo setting.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Indicán/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/farmacología , Humanos , Indicán/administración & dosificación , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Vascular calcification is frequent in patients with chronic kidney disease. Osteoprotegerin (OPG, a soluble factor which blocks osteoclast differentiation) has recently been implicated in the genesis of vascular calcification. Given that OPG can bind the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we hypothesized that the TRAIL protein is involved in the formation of vascular calcification both in vitro and in vivo. Using an immunohistochemical approach, we evaluated TRAIL and OPG expression on aortic valves slides from non-uremic and uremic wild type and apolipoprotein knockout (Apo E(-/-) ) mice. We also tested the in vitro effects of TRAIL on cultured primary human vascular smooth muscle cells (hVSMC). We further assayed serum soluble TRAIL (sTRAIL) levels in hemodialysis patients and correlated them with vascular calcification scores. Our results demonstrated that: (i) TRAIL and OPG were expressed inside the atheroma plaque in non-uremic ApoE(-/-) mice, but not in wild type mice; and (ii) uremia enhanced the expression levels. TRAIL enhanced the phosphate-induced mineralization of hVSMCs in a dose-dependent manner. In clinical terms, we demonstrated that sTRAIL is depressed in the sera of hemodialysis patients, but was not correlated with vascular calcification. Our results suggest that TRAIL may be involved in the formation of vascular calcification in certain experimental settings; however, its role in chronic kidney disease patients requires further evaluation.

Asunto(s)

Calcinosis/fisiopatología , Enfermedades Renales/fisiopatología , Osteoprotegerina/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Anciano , Animales , Apolipoproteínas E/genética , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedades Renales/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Osteoprotegerina/sangre , Osteoprotegerina/genética , Placa Aterosclerótica/patología , Diálisis Renal/métodos , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Uremia/fisiopatología

RESUMEN

Chronic kidney disease (CKD) is frequently complicated by arterial calcification. The latter is part of the associated mineral and bone disorder (CKD-MBD). Hypercalcemia and hyperphosphatemia have long been known to play a major role in the occurrence of vascular and other soft tissue calcification in patients with CKD, together with endocrine disturbances including vitamin D, parathyroid hormone, fibroblast growth factor-23, and klotho. In addition, many other systemic and local promoters, including inflammation and uremic toxins, contribute to the occurrence of vascular calcification, despite a powerful defense system made up of systemic and local inhibitors, as demonstrated in elegant experimental studies done in vitro and in vivo. Most importantly, several reports have shown that both hyperphosphatemia and hypophosphatemia, and to a lesser degree hypercalcemia and hypocalcemia, are associated with an increased relative risk of mortality in patients with CKD. However, all these reports were observational in nature and must therefore be considered as hypothesis generating. It remains to be demonstrated in prospective randomized trials whether normalization of serum phosphorus and/or calcium leads to better patient outcome. In order to improve outcome in patients with CKD-MBD, early medical intervention is of utmost importance.

Asunto(s)

Calcio/metabolismo , Enfermedades Renales/metabolismo , Fosfatos/metabolismo , Calcinosis/etiología , Humanos , Enfermedades Renales/complicaciones , Enfermedades Renales/mortalidad , Enfermedades Renales/patología , Enfermedades Vasculares/etiología , Enfermedades Vasculares/patología

RESUMEN

In this work, we investigated the effect of inorganic phosphate (Pi) on the differentiation of monocyte/macrophage precursors into an "osteoclastic" phenotype, and we delineated the molecular mechanisms which could be involved in this phenomenon. This was achieved by stimulating human peripheral blood monocytic cells and RAW 264.7 monocyte-macrophage precursor cells to differentiate into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). RANKL has been previously reported to stimulate the signaling kinases ERK 1/2, p38, Akt, JNK, and the DNA-binding activity of the transcription factors AP-1 and NF-kappaB. Increase in extracellular Pi concentration (1.5-4.5 mM) dose-dependently inhibits both osteoclastic differentiation and bone resorption activity induced by RANKL and M-CSF. Pi was found to specifically inhibit the RANKL-induced JNK and Akt activation, while RANKL-induced p38 and ERK 1/2 phosphorylation were not significantly affected. Moreover, we found that Pi significantly reduced the RANKL-stimulated DNA-binding activity of NF-kappaB. The effects of Pi on osteoclast differentiation and DNA-binding activity of NF-kappaB were prevented by Foscarnet, a sodium-phosphate cotransport inhibitor, suggesting that the effects of Pi occur subsequently to its intake. These results demonstrate that Pi downregulates the differentiation of osteoclasts via a negative feedback exerted on RANK-RANKL signaling.

Asunto(s)

Espacio Extracelular/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Fosfatos/farmacología , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Resorción Ósea , Diferenciación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Foscarnet/farmacología , Humanos , Isoenzimas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Osteoclastos/citología , Osteoclastos/enzimología , Osteogénesis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ligando RANK/farmacología , Proteínas Cotransportadoras de Sodio-Fosfato/antagonistas & inhibidores , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Fosfatasa Ácida Tartratorresistente , Factor de Transcripción AP-1/metabolismo