RESUMO
Microplastics (MPs) and nanoplastics are small synthetic organic polymer particles (<5 mm and <1 µm, respectively) that originate directly from plastic compounds or result from the degradation of plastic. These particles are a global concern because they are widely distributed in water, air, food, and soil, and recent scientific evidence has linked MPs to negative biological effects. Although these particles are difficult to detect in humans, MPs have been identified in different biological fluids and tissues, such as the placenta, lung, intestines, liver, blood, urine, and kidneys. Human exposure to MPs can occur by ingestion, inhalation, or dermal contact, potentially causing metabolic alterations. Data from experimental and clinical studies have revealed that the ability of MPs to promote inflammation, oxidative stress, and organ dysfunction and negatively affect clinical outcomes is associated with their accumulation in body fluids and tissues. Although evidence of the putative action of MPs in the human kidney is still scarce, there is growing interest in studying MPs in this organ. In addition, chronic kidney disease requires investigation because this condition is potentially prone to MP accumulation. The purpose of the present article is (i) to review the general aspects of MP generation, available analytic methods for identification, and the main known biological toxic effects; and (ii) to describe and critically analyze key experimental and clinical studies that support a role of MPs in kidney disease.
Assuntos
Rim , Microplásticos , Humanos , Microplásticos/toxicidade , Rim/efeitos dos fármacos , Rim/metabolismo , Animais , Exposição Ambiental/efeitos adversos , Estresse Oxidativo/efeitos dos fármacosRESUMO
Within the cardiovascular system, the protein vasorin (Vasn) is predominantly expressed by vascular smooth muscle cells (VSMCs) in the coronary arteries and the aorta. Vasn knockout (Vasn-/- ) mice die within 3 weeks of birth. In the present study, we investigated the role of vascular Vasn expression on vascular function. We used inducible Vasn knockout mice (VasnCRE-ERT KO and VasnSMMHC-CRE-ERT2 KO , in which respectively all cells or SMCs only are targeted) to analyze the consequences of total or selective Vasn loss on vascular function. Furthermore, in vivo effects were investigated in vitro using human VSMCs. The death of VasnCRE-ERT KO mice 21 days after tamoxifen injection was concomitant with decreases in blood pressure, angiotensin II levels, and vessel contractibility to phenylephrine. The VasnSMMHC-CRE-ERT2 KO mice displayed concomitant changes in vessel contractibility in response to phenylephrine and angiotensin II levels. In vitro, VASN deficiency was associated with a shift toward the SMC contractile phenotype, an increase in basal intracellular Ca2+ levels, and a decrease in the SMCs' ability to generate a calcium signal in response to carbachol or phenylephrine. Additionally, impaired endothelium-dependent relaxation (due to changes in nitric oxide signaling) was observed in all Vasn knockout mice models. Our present findings highlight the role played by Vasn SMC expression in the maintenance of vascular functions. The mechanistic experiments suggested that these effects are mediated by SMC phenotype switching and changes in intracellular calcium homeostasis, angiotensin II levels, and NO signaling.
Assuntos
Angiotensina II , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/metabolismo , Músculo Liso Vascular , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Carbacol , Humanos , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Fenilefrina/metabolismo , TamoxifenoRESUMO
Vascular dysfunction is an essential element found in many cardiovascular pathologies and in pathologies that have a cardiovascular impact such as chronic kidney disease (CKD). Alteration of vasomotricity is due to an imbalance between the production of relaxing and contracting factors. In addition to becoming a determining factor in pathophysiological alterations, vascular dysfunction constitutes the first step in the development of atherosclerosis plaques or vascular calcifications. In patients with CKD, alteration of vasomotricity tends to emerge as being a new, less conventional, risk factor. CKD is characterized by the accumulation of uremic toxins (UTs) such as phosphate, para-cresyl sulfate, indoxyl sulfate, and FGF23 and, consequently, the deleterious role of UTs on vascular dysfunction has been explored. This accumulation of UTs is associated with systemic alterations including inflammation, oxidative stress, and the decrease of nitric oxide production. The present review proposes to summarize our current knowledge of the mechanisms by which UTs induce vascular dysfunction.
Assuntos
Vasos Sanguíneos/metabolismo , Cresóis/sangue , Indicã/sangue , Insuficiência Renal Crônica/complicações , Ésteres do Ácido Sulfúrico/sangue , Uremia/complicações , Doenças Vasculares/etiologia , Animais , Vasos Sanguíneos/fisiopatologia , Fator de Crescimento de Fibroblastos 23 , Humanos , Mediadores da Inflamação/metabolismo , Estresse Oxidativo , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/fisiopatologia , Insuficiência Renal Crônica/terapia , Uremia/sangue , Uremia/fisiopatologia , Uremia/terapia , Doenças Vasculares/sangue , Doenças Vasculares/fisiopatologia , Doenças Vasculares/prevenção & controleRESUMO
Sirtuin of type 1 (Sirt1), a class III HDAC, is known to be involved in the regulation of differentiation of skeletal stem cells (SSCs) into osteoblasts and adipocytes. In caloric restriction, it has been shown that the expression and activity of Sirt1 is a tissue-dependent regulation. However, at present, no study has focused on the link between Sirt1, bone marrow adiposity (BMA) and osteoporosis related to anorexia nervosa (AN). Thus, the aims of this work were to (i) determine BMA and bone changes in a mouse model replicating the phenotypes of AN (separation-based anorexia model (SBA)); (ii) determine the expression of Sirt1 in bone marrow stromal cells (BMSCs) extracted from these mice and identify their differentiation capacities; (iii) study the effects of pharmacological activation and inhibition of Sirt1 on the osteoblastogenesis and adipogenesis of these cells and (iiii) delineate the molecular mechanism by which Sirt1 could regulate osteogenesis in an SBA model. Our results demonstrated that SBA protocol induces an increase in BMA and alteration of bone architecture. In addition, BMSCs from restricted mice present a down-regulation of Sirt1 which is accompanied by an increase in adipogenesis at expense of osteogenesis. After a 10-day organotypic culture, tibias from SBA mice displayed low levels of Sirt1 mRNA which are restored by resveratrol treatment. Interestingly, this recovery of Sirt1 levels also returned the BMA, BV/TV and Tb.Th in cultured tibias from SBA mice to normal levels. In contrast of down-regulation of Sirt1 expression induced by sirtinol treatment, stimulation of Sirt1 expression by resveratrol lead to a decrease in adipogenesis and increase in osteogenesis. Finally, to investigate the molecular mechanisms by which Sirt1 could regulate osteogenesis in the SBA model, the acetylation levels of Runx2 and Foxo1 transcription factors were determined. Our data show that this chronic energy deficiency in female mice causes a decrease in BMSC activity, resulting in critical changes to Runx2 and Foxo1 acetylation levels and thus to their activity. Altogether, these data suggest that Sirt1 could be considered as a potential therapeutic target in osteoporosis related to AN.
Assuntos
Medula Óssea , Sirtuína 1 , Adipogenia , Adiposidade , Animais , Medula Óssea/metabolismo , Diferenciação Celular , Feminino , Camundongos , Osteoblastos/metabolismo , Osteogênese , Sirtuína 1/metabolismoAssuntos
Regulação da Expressão Gênica/fisiologia , Macrófagos/metabolismo , MicroRNAs/metabolismo , Monócitos/metabolismo , Animais , Proteínas de Transporte/biossíntese , Proteínas de Drosophila/biossíntese , Genômica , Macrófagos/citologia , Camundongos , Monócitos/citologia , Proteína-Arginina N-Metiltransferases/biossíntese , Células RAW 264.7 , Enzimas de Conjugação de Ubiquitina/biossínteseRESUMO
RATIONALE: Vascular calcification is a process similar to bone formation leading to an inappropriate deposition of calcium phosphate minerals in advanced atherosclerotic plaques. Monocyte-derived macrophages, located in atherosclerotic lesions and presenting heterogeneous phenotypes, from classical proinflammatory M1 to alternative anti-inflammatory M2 macrophages, could potentially display osteoclast-like functions. OBJECTIVE: To characterize the phenotype of macrophages located in areas surrounding the calcium deposits in human atherosclerotic plaques. METHODS AND RESULTS: Macrophages near calcium deposits display an alternative phenotype being both CD68 and mannose receptor-positive, expressing carbonic anhydrase type II, but relatively low levels of cathepsin K. In vitro interleukin-4-polarization of human primary monocytes into macrophages results in lower expression and activity of cathepsin K compared with resting unpolarized macrophages. Moreover, interleukin-4 polarization lowers expression levels of the osteoclast transcriptional activator nuclear factor of activated T cells type c-1, associated with increased gene promoter levels of the transcriptional repression mark H3K27me3 (histone 3 lysine 27 trimethylation). Despite higher expression of the receptor activator of nuclear factor κB receptor, receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor induction of nuclear factor of activated T cells type c-1 and cathepsin K expression is defective in these macrophages because of reduced Erk/c-fos-mediated downstream signaling resulting in impaired bone resorption capacity. CONCLUSIONS: These results indicate that macrophages surrounding calcium deposits in human atherosclerotic plaques are phenotypically defective being unable to resorb calcification.
Assuntos
Reabsorção Óssea/metabolismo , Macrófagos/metabolismo , Osteoclastos/metabolismo , Placa Aterosclerótica/metabolismo , Ligante RANK/metabolismo , Calcificação Vascular/metabolismo , Reabsorção Óssea/patologia , Células Cultivadas , Humanos , Microdissecção e Captura a Laser/métodos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Macrófagos/patologia , Osteoclastos/patologia , Placa Aterosclerótica/patologia , Calcificação Vascular/patologiaRESUMO
N-methyl-2-pyridone-5-carboxamide (2PY, a major metabolite of nicotinamide, NAM) was recently identified as a uremic toxin. Recent interventional trials using NAM to treat high levels of phosphorus in end-stage renal disease have highlighted new potential uremic toxicities of 2PY. In the context of uremia, the accumulation of 2PY could be harmful-perhaps by inhibiting poly (ADP-ribose) polymerase-1 activity. Here, we review recently published data on 2PY's metabolism and toxicological profile.
Assuntos
Niacinamida/análogos & derivados , Niacinamida/metabolismo , Toxinas Biológicas/metabolismo , Uremia/metabolismo , Animais , Humanos , Insuficiência Renal Crônica/metabolismoRESUMO
Vascular calcification (VC) is prevalent in patients suffering from chronic kidney disease (CKD). High phosphate levels promote VC by inducing abnormalities in mineral and bone metabolism. Previously, we demonstrated that magnesium (Mg(2+)) prevents inorganic phosphate- (Pi-) induced VC in human aortic vascular smooth muscle cells (HAVSMC). As microRNAs (miR) modulate gene expression, we investigated the role of miR-29b, -30b, -125b, -133a, -143, and -204 in the protective effect of Mg(2+) on VC. HAVSMC were cultured in the presence of 3 mM Pi with or without 2 mM Mg(2+) chloride. Total RNA was extracted after 4 h, 24 h, day 3, day 7, and day 10. miR-30b, -133a, and -143 were downregulated during the time course of Pi-induced VC, whereas the addition of Mg(2+) restored (miR-30b) or improved (miR-133a, miR-143) their expression. The expression of specific targets Smad1 and Osterix was significantly increased in the presence of Pi and restored by coincubation with Mg(2+). As miR-30b, miR-133a, and miR-143 are negatively regulated by Pi and restored by Mg(2+) with a congruent modulation of their known targets Runx2, Smad1, and Osterix, our results provide a potential mechanistic explanation of the observed upregulation of these master switches of osteogenesis during the course of VC.
Assuntos
Magnésio/administração & dosagem , MicroRNAs/metabolismo , Fosfatos/administração & dosagem , Proteína Smad1/metabolismo , Fatores de Transcrição/metabolismo , Calcificação Vascular/metabolismo , Cálcio/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fator de Transcrição Sp7RESUMO
Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a common complication of CKD, and uremic toxins have been shown to be instrumental in this process. We have previously shown that miR-223 is increased in smooth muscle cells subjected to the uremic toxin inorganic phosphate (Pi). In the present study we investigated the influence of this miRNA in osteoclastogenesis in order to elucidate its role in the course of CKD-MBD. RT-qPCR demonstrated that high Pi concentration decreased miR-223 expression in differentiated RAW 264.7 cells. Up- and down-regulation of miR-223 was performed using specific pre-miR and anti-miR-223. Differentiation of monocyte/macrophage precursors was assessed by using RAW 264.7 cells and peripheral blood mononuclear cells (PBMC). TRAP activity and bone resorption were used to measure osteoclast activity. Pi induced a marked decrease in osteoclastogenesis in RAW cells and miR-223 levels were concomitantly decreased. Anti-miR-223 treatment inhibited osteoclastogenesis in the same way as Pi. In contrast, overexpression of miR-223 triggered differentiation, as reflected by TRAP activity. We showed that miR-223 affected the expression of its target genes NFIA and RhoB, but also osteoclast marker genes and the Akt signalling pathway, which induces osteoclastogenesis. These results were confirmed by measuring bone resorption activity of human PBMC differentiated into osteoclasts. We thus demonstrate a role of miR-223 in osteoclast differentiation, with rational grounds to use deregulation of this miRNA to selectively increase osteoclast-like activity in calcified vessels of CKD-MBD. This approach could alleviate vascular calcification without altering bone structure.
RESUMO
Chronic kidney disease (CKD) is generally associated with disturbances of mineral and bone metabolism. They contribute to the development of vascular calcification (VC), a strong, independent predictor of cardiovascular risk. Pyrophosphate (PPi), an endogenous inhibitor of hydroxyapatite formation, has been shown to slow the progression of VC in uremic animals. Since in patients with CKD treatment is usually initiated for already existing calcifications, we aimed to compare the efficacy of PPi therapy with that of the phosphate binder sevelamer, using a uremic apolipoprotein-E knockout mouse model with advanced VCs. After CKD creation or sham surgery, 12-week-old female mice were randomized to one sham group and four CKD groups (n = 18-19/group). Treatment was initiated 8 weeks after left nephrectomy allowing prior VC development. Uremic groups received either intraperitoneal PPi (high dose, 1.65 mg/kg or low dose, 0.33 mg/kg per day), oral sevelamer (3 % in diet), or placebo treatment for 8 weeks. Both intima and media calcifications worsened with time in placebo-treated CKD mice, based on both quantitative image analysis and biochemical measurements. Progression of calcification between 8 and 16 weeks was entirely halted by PPi treatment, as it was by sevelamer treatment. PPi did not induce consistent bone histomorphometry changes. Finally, the beneficial vascular action of PPi probably involved mechanisms different from that of sevelamer. Further studies are needed to gain more precise insight into underlying mechanisms and to see whether PPi administration may also be useful in patients with CKD and VC.
Assuntos
Difosfatos/administração & dosagem , Calcificação Vascular/patologia , Animais , Apolipoproteínas E/deficiência , Modelos Animais de Doenças , Progressão da Doença , Infusões Parenterais , Camundongos , Camundongos Knockout , Insuficiência Renal Crônica/complicações , Uremia/complicações , Calcificação Vascular/prevenção & controleRESUMO
BACKGROUND: Cardiovascular disease including vascular calcification (VC) remains the leading cause of death in patients suffering from chronic kidney disease (CKD). The process of VC seems likely to be a tightly regulated process where vascular smooth muscle cells are playing a key role rather than just a mere passive precipitation of calcium phosphate. Characterisation of the chemical and crystalline structure of VC was mainly led in patients or animal models with CKD. Likewise, Mg2+ was found to be protective in living cells although a potential role for Mg2+ could not be excluded on crystal formation and precipitation. In this study, the crystal formation and the role of Mg2+ were investigated in an in vitro model of primary human aortic vascular smooth muscle cells (HAVSMC) with physical techniques. METHODOLOGY/PRINCIPAL FINDINGS: In HAVSMC incubated with increased Ca x Pi medium, only calcium phosphate apatite crystals (CPA) were detected by Micro-Fourier Transform InfraRed spectroscopy (µFTIR) and Field Effect Scanning Electron Microscope (FE-SEM) and Energy Dispersive X-ray spectrometry (EDX) at the cell layer level. Supplementation with Mg2+ did not alter the crystal composition or structure. The crystal deposition was preferentially positioned near or directly on cells as pictured by FE-SEM observations and EDX measurements. Large µFTIR maps revealed spots of CPA crystals that were associated to the cellular layout. This qualitative analysis suggests a potential beneficial effect of Mg2+ at 5 mM in noticeably reducing the number and intensities of CPA µFTIR spots. CONCLUSIONS/SIGNIFICANCE: For the first time in a model of HAVSMC, induced calcification led to the formation of the sole CPA crystals. Our data seems to exclude a physicochemical role of Mg2+ in altering the CPA crystal growth, composition or structure. Furthermore, Mg2+ beneficial role in attenuating VC should be linked to an active cellular role.
Assuntos
Aorta/metabolismo , Fosfatos de Cálcio/metabolismo , Magnésio/metabolismo , Miócitos de Músculo Liso/metabolismo , Calcificação Vascular/metabolismo , Aorta/patologia , Células Cultivadas , Feminino , Humanos , Masculino , Miócitos de Músculo Liso/patologia , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Calcificação Vascular/patologiaRESUMO
BACKGROUND AND AIMS: Sclerostin is a circulating inhibitor of the Wnt/ß-catenin pathway and may have a role in chronic kidney disease (CKD)-mineral and bone disorder. Blood sclerostin levels are known to be elevated in patients undergoing maintenance dialysis. The aims of the present study were to evaluate sclerostin levels in patients at different CKD stages and study potential associations between sclerostin levels and (i) biochemical parameters that are disturbed in CKD, (ii) markers of vascular disease and (iii) mortality. METHODS: One hundred and forty patients at CKD stages 2-5D were included in the present study. Routine clinical biochemistry tests and assays for sclerostin, protein-bound uremic toxins (indoxylsulphate [IS] and p-cresyl sulphate [PCS]) and the toxin ß2 microglobulin (ß2M) were performed. Aortic and coronary calcification and arterial stiffness were assessed by multislice spiral computed tomography and pulse wave velocity measurements. The enrolled patients were prospectively monitored for mortality. RESULTS: Sclerostin levels were found to be elevated in CKD patients (especially those on hemodialysis). Furthermore, sclerostin levels were positively correlated with inflammation markers, phosphate, fibroblast growth factor 23, IS, PCS, ß2M and arterial stiffness. A multivariate linear regression analysis indicated that sclerostin levels were independently associated with IS, PCS and ß2M levels. Elevated serum sclerostin appeared to be associated with mortality (independently of age and inflammation). However, this association disappeared after adjustment for a propensity score including age, phosphate, interleukin-6, CKD stage and PCS. CONCLUSION: Our results indicate that sclerostin levels are elevated in CKD patients and are associated with inflammation, vascular lesions, uremia and (potentially) mortality.
Assuntos
Proteínas Morfogenéticas Ósseas/sangue , Insuficiência Renal Crônica/sangue , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Aortografia , Arteriosclerose/diagnóstico por imagem , Arteriosclerose/etiologia , Arteriosclerose/fisiopatologia , Biomarcadores , Calcinose/diagnóstico por imagem , Calcinose/etiologia , Calcinose/fisiopatologia , Cresóis/sangue , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Seguimentos , Marcadores Genéticos , Humanos , Indicã/sangue , Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Análise de Onda de Pulso , Insuficiência Renal Crônica/complicações , Ésteres do Ácido Sulfúrico/sangue , Análise de Sobrevida , Tomografia Computadorizada Espiral , Uremia/sangue , Rigidez Vascular , Microglobulina beta-2/análiseRESUMO
Vascular calcification (VC) is a risk factor for cardiovascular mortality in the setting of chronic kidney disease (CKD). Pyrophosphate (PPi), an endogenous molecule that inhibits hydroxyapatite crystal formation, has been shown to prevent the development of VC in animal models of CKD. However, the possibility of harmful effects of exogenous administration of PPi on bone requires further investigation. To this end, we examined by histomorphometry the bone of CKD mice after intraperitoneal PPi administration. After CKD creation or sham surgery, 10-week-old female apolipoprotein-E knockout (apoE(-/-)) mice were randomized to one non-CKD group or 4 CKD groups (n = 10-35/group) treated with placebo or three distinct doses of PPi, and fed with standard diet. Eight weeks later, the animals were killed. Serum and femurs were sampled. Femurs were processed for bone histomorphometry. Placebo-treated CKD mice had significantly higher values of osteoid volume, osteoid surface and bone formation rate than sham-placebo mice with normal renal function. Slightly higher osteoid values were observed in CKD mice in response to very low PPi dose (OV/BV, O.Th and ObS/BS) and, for one parameter measured, to high PPi dose (O.Th), compared to placebo-treated CKD mice. Treatment with PPi did not modify any other structural parameters. Mineral apposition rates, and other parameters of bone formation and resorption were not significantly different among the treated animal groups or control CKD placebo group. In conclusion, PPi does not appear to be deleterious to bone tissue in apoE(-/-) mice with CKD, although a possible stimulatory PPi effect on osteoid formation may be worth further investigation.
Assuntos
Apolipoproteínas E/genética , Densidade Óssea/efeitos dos fármacos , Soluções para Diálise/farmacologia , Difosfatos/farmacologia , Fêmur/metabolismo , Diálise Peritoneal/métodos , Insuficiência Renal Crônica/terapia , Calcificação Vascular/prevenção & controle , Animais , Densidade Óssea/genética , Feminino , Fêmur/patologia , Camundongos , Camundongos Knockout , Diálise Peritoneal/efeitos adversos , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Calcificação Vascular/etiologia , Calcificação Vascular/genética , Calcificação Vascular/metabolismo , Calcificação Vascular/patologiaRESUMO
Chronic kidney disease (CKD) is associated with vascular calcifications and atherosclerosis. There is a need for novel predictors to allow earlier diagnosis of these disorders, predict disease progression, and improve assessment of treatment response. We focused on microRNAs since they are implicated in a variety of cellular functions in cardiovascular pathology. We examined changes of microRNA expression in aortas of CKD and non-CKD wild type mice and apolipoprotein E knock-out mice, respectively. Both vascular smooth muscle-specific miR-143 and miR-145 expressions were decreased in states of atherosclerosis and/or CKD or both, and the expression level of protein target Myocardin was increased. The inflammatory miR-223 was increased in more advanced stages of CKD, and specific protein targets NFI-A and GLUT-4 were dramatically decreased. Expression of miR-126 was markedly increased and expression of protein targets VCAM-1 and SDF-1 was altered during the course of CKD. The drug sevelamer, commonly used in CKD, corrected partially these changes in microRNA expression, suggesting a direct link between the observed microRNA alterations and uremic vascular toxicity. Finally, miR-126, -143 and -223 expression levels were deregulated in murine serum during the course of experimental CKD. In conclusion, these miRNAs could have role(s) in CKD vascular remodeling and may therefore represent useful targets to prevent or treat complications of CKD.
Assuntos
Aorta/metabolismo , Aterosclerose/genética , MicroRNAs/genética , Insuficiência Renal Crônica/genética , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/complicações , Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poliaminas/farmacologia , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Sevelamer , Transativadores/genética , Transativadores/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: Vascular calcification (VC) is prevalent in patients suffering from chronic kidney disease. Factors promoting calcification include abnormalities in mineral metabolism, particularly high phosphate levels. Inorganic phosphate (Pi) is a classical inducer of in vitro VC. Recently, an inverse relationship between serum magnesium concentrations and VC has been reported. The present study aimed to investigate the effects of magnesium on Pi-induced VC at the cellular level using primary HAVSMC. METHODS: Alive and fixed HAVSMC were assessed during 14 days in the presence of Pi with increasing concentrations of magnesium (Mg(2+)) chloride. Mineralization was measured using quantification of calcium, von Kossa and alizarin red stainings. Cell viability and secretion of classical VC markers were also assessed using adequate tests. Involvement of transient receptor potential melastatin (TRPM) 7 was assessed using 2-aminoethoxy-diphenylborate (2-APB) inhibitor. RESULTS: Co-incubation with Mg(2+) significantly decreased Pi-induced VC in live HAVSMC, no effect was found in fixed cells. At potent concentrations in Pi-induced HAVSMC, Mg(2+) significantly improved cell viability and restored to basal level increased secretions of osteocalcin and matrix gla protein, whereas a decrease in osteopontin secretion was partially restored. The block of TRPM7 with 2-APB at 10(-4) M led to the inefficiency of Mg(2+) to prevent VC. CONCLUSIONS: Increasing Mg(2+) concentrations significantly reduced VC, improved cell viability and modulated secretion of VC markers during cell-mediated matrix mineralization clearly pointing to a cellular role for Mg(2+) and 2-APB further involved TRPM7 and a potential Mg(2+) entry to exert its effects. Further investigations are needed to shed light on additional cellular mechanism(s) by which Mg(2+) is able to prevent VC.
Assuntos
Aorta/metabolismo , Magnésio/farmacologia , Minerais/metabolismo , Músculo Liso Vascular/metabolismo , Fosfatos/metabolismo , Calcificação Vascular/patologia , Aorta/citologia , Aorta/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Calcificação Vascular/metabolismoRESUMO
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.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Indicã/farmacologia , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Animais , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , MAP Quinase Quinase 4/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Osteoclastos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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.
Assuntos
Proliferação de Células/efeitos dos fármacos , Indicã/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Aorta/efeitos dos fármacos , Aorta/metabolismo , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Humanos , Indicã/administração & dosagem , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: The high rate of cardiovascular mortality in patients with end-stage renal disease (ESRD) is a significant barrier to improved life expectancy. Unique in this population is the marked development and aggressive worsening of vascular calcification (VC). Pyrophosphate (PPi), an endogenous molecule, appears to naturally inhibit soft tissue calcification, but may be depressed in chronic kidney disease (CKD) and ESRD. Although once thought to be a promising therapeutic, PPi's very short half-life in circulation curtailed earlier studies. We tested the possibility that a slow, continuous entry of PPi into the circulation and prevention of VC might be achieved by daily peritoneal dialysis (PD). METHODS: Pharmacokinetic studies were first carried out in rats with renal impairment resulting from a 5/6 nephrectomy. Efficacy studies were then performed in the apolipoprotein E gene knockout mouse model overlaid with CKD. PPi was delivered by means of a permanent peritoneal catheter in a solution simulating PD, but without the timed removal of spent dialysate. von Kossa's staining followed by semiquantitative morphological image processing, with separation of inside (intimal) and outside (presumed medial) lesions, was used to determine aortic root calcification. RESULTS: In comparison to an intravenous bolus, delivery of PPi in a PD solution resulted in a slower, extended delivery over >4 h. Next, the efficacy studies showed that a 6-day/week PD-simulated administration of PPi resulted in a dose-dependent inhibition of aortic calcification in both intimal and medial lesions. A dose-response effect on total aortic calcification was also documented, with a full inhibition seen at the highest dose. A limited peritoneal catheter-related inflammation was observed, as expected, and included the placebo-treated control groups. This inflammatory response could have masked a lower level PPi-specific adverse effect, but none was observed. CONCLUSIONS: Our findings suggest potential for PPi, administered during PD, to prevent the development of VC and to potentially extend the life of ESRD patients.
Assuntos
Apolipoproteínas E/fisiologia , Soluções para Diálise/uso terapêutico , Difosfatos/administração & dosagem , Diálise Peritoneal/efeitos adversos , Insuficiência Renal/terapia , Calcificação Vascular/etiologia , Calcificação Vascular/prevenção & controle , Animais , Cálcio/metabolismo , Soluções para Diálise/farmacocinética , Difosfatos/farmacocinética , Feminino , Meia-Vida , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Insuficiência Renal/complicações , Distribuição Tecidual , Uremia/tratamento farmacológico , Uremia/etiologiaRESUMO
Chronic kidney disease is considered a major cause of cardiovascular risk and non-traditional risk factors remain largely unknown. The in vitro toxicity of 10 guanidino compounds (GCs) was evaluated via a standardized approach on different cell systems of relevance in cardiovascular disease. The parameters evaluated were production of reactive oxygen species, expression of surface molecules, cell proliferation, cytotoxicity and calcification. Several GCs had a stimulatory effect on monocytes and granulocytes (SDMA, creatine and guanidinobutyric acid (GBA)). Some GCs (guandine (G), guanidinosuccinic acid (GSA) and SDMA) inhibited endothelial cell proliferation or reduced calcification in osteoblast-like human VSMC (ADMA, GSA and SDMA). Stimulation of osteoclastogenesis could be demonstrated for ADMA, G, guanidinoacetic acid and GBA in a RAW264.7 cell line. No compounds were cytotoxic to AoSMC or endothelial cells, nor influenced their viability. GCs, especially SDMA, likely contribute to cardiovascular complications in uremia, mainly those related to microinflammation and leukocyte activation.
Assuntos
Doenças Cardiovasculares , Guanidinas , Falência Renal Crônica/complicações , Insuficiência Renal Crônica/complicações , Calcinose/metabolismo , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Guanidinas/efeitos adversos , Guanidinas/toxicidade , Humanos , Falência Renal Crônica/metabolismo , Leucócitos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos/análise , Receptores de Antígenos/efeitos dos fármacos , Insuficiência Renal Crônica/metabolismo , Risco , Uremia/complicações , Uremia/metabolismoRESUMO
The role of OxLDL in the generation and progression of atherosclerosis is well admitted. In addition, it is well known that atherosclerosis is often accompanied by perturbations in bone remodeling, resulting in osteoporosis. In the current studies, the effect of Cu(2+)-oxidized LDL (OxLDL) on RANKL-induced RAW264.7 mouse monocytes-macrophages differentiation to osteoclasts and on RANKL signaling pathway was investigated. OxLDL, within the range of 10-50 microg protein/ml, prevented RANKL-induced generation of multinucleated osteoclast-like cells and RANKL-induced tartrate resistant acid phosphatase (TRAP) activity. OxLDL also prevented the RANKL-induced phosphorylation of ERK, p38 and JNK kinases, together with the RANKL-induced DNA binding activities of NFkappaB and NFAT transcription factors. Concomitantly, OxLDL enhanced RANKL-induced generation of reactive oxygen species in a dose-dependent manner. The antioxidant glutathione (GSH) prevented whereas the prooxidant compound buthionine-sulfoximine (BSO) enhanced the effect of OxLDL on RANKL-induced oxidative stress and RANKL-induced differentiation. Finally, OxLDL also prevented RANKL-induced TRAP activity and RANKL-induced bone resorbing activity of human peripheral blood mononuclear cells. These results demonstrate that OxLDL, by generation of an intracellular oxidative stress, prevents the differentiation of osteoclasts by inhibition of RANKL signaling pathway. This might be related to the fact that atherosclerosis is accompanied by perturbations in bone and vascular remodeling, leading to osteoporosis and vascular calcification.