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Resumen: Antecedentes y objetivos: Los pacientes con insuficiencia renal crónica (IRC) corren mayor riesgo de desarrollar enfermedad cardiovascular. En los pacientes con IRC, los mecanismos implicados en la disfunción endotelial y el papel de los diferentes fármacos utilizados en estos pacientes no se conocen por completo. El objetivo de este artículo es analizar el efecto de las estatinas y los antiagregantes plaquetarios (AP) sobre las microvesículas endoteliales (MVE) y otros marcadores de la disfunción endotelial. Enfoque experimental: Estudio transversal con 41 pacientes con IRC 3b-4 y 8 voluntarios sanos. Se cuantificaron los niveles de MVE, factor de crecimiento vascular endotelial (FCVE) y productos avanzados de oxidación de proteínas (AOPP, por sus siglas en inglés) en la circulación y se evaluó la correlación con diferentes variables de comorbilidad y estrategias terapéuticas. Resultados: Las MVE aumentaron en pacientes con IRC al comparar los niveles con los controles (171,1 frente a 68,3/μl; p < 0,001). Se observó una correlación negativa entre la edad y las MVE. Las estatinas y los AP se asociaron con una reducción de los niveles de MVE y FCVE, independientemente de los niveles séricos de colesterol total (CT). Los niveles de AOPP y FCVE no fueron diferentes entre los pacientes con IRC y los controles. Conclusión: La IRC se asocia con un cambio de los niveles de MVE, FCVE y AOPP. El tratamiento con estatinas y AP normaliza estos valores a casi los observados en los controles y este efecto es independiente del nivel de CT predominante. Estos hallazgos explican la existencia de los efectos pleiotrópicos de las estatinas y los AP que merecen estudios adicionales. Abstract: Backgrounds and purposes: Patients with chronic kidney disease (CKD) have higher risk of developing cardiovascular disease. In CKD patients the mechanisms involved in, endothelial damage and the role of different drugs used on these patients are not completely understood. The aim of this work is to analyze the effect of statins and platelet antiaggregant (PA) on endothelial microvesicles (EMVs) and other markers of endothelial dysfunction. Experimental approach: Cross-sectional study of 41 patients with CKD 3b-4 and 8 healthy volunteers. Circulating levels of EMVs, vascular endothelial growth factor (VEGF), and advance oxidized protein products (AOPPS) were quantified and the correlation with different comorbidity variables and therapeutic strategies were evaluated. Results: EMVs are increased in CKD patients as compared with controls (171.1 vs. 68.3/μl, P

Although magnesium has been shown to prevent vascular calcification in vitro , controlled in vivo studies in uremic animal models are limited. To determine whether dietary magnesium supplementation protects against the development of vascular calcification, 5/6 nephrectomized Wistar rats were fed diets with different magnesium content increasing from 0.1 to 1.1%. In one study we analyzed bone specimens from rats fed 0.1%, 0.3%, and 0.6% magnesium diets, and in another study we evaluated the effect of intraperitoneal magnesium on vascular calcification in 5/6 nephrectomized rats. The effects of magnesium on established vascular calcification were also evaluated in uremic rats fed on diets with either normal (0.1%) or moderately increased magnesium (0.6%) content. The increase in dietary magnesium resulted in a marked reduction in vascular calcification, together with improved mineral metabolism and renal function. Moderately elevated dietary magnesium (0.3%), but not high dietary magnesium (0.6%), improved bone homeostasis as compared to basal dietary magnesium (0.1%). Results of our study also suggested that the protective effect of magnesium on vascular calcification was not limited to its action as an intestinal phosphate binder since magnesium administered intraperitoneally also decreased vascular calcification. Oral magnesium supplementation also reduced blood pressure in uremic rats, and in vitro medium magnesium decreased BMP-2 and p65–NF-κB in TNF-α–treated human umbilical vein endothelial cells. Finally, in uremic rats with established vascular calcification, increasing dietary magnesium from 0.1% magnesium to 0.6% reduced the mortality rate from 52% to 28%, which was associated with reduced vascular calcification. Thus, increasing dietary magnesium reduced both vascular calcification and mortality in uremic rats.

Renal dysfunction is closely associated with endothelial damage leading to cardiovascular disease. However, the extent to which endothelial damage induced by uremia is modulated by aging is poorly known. Aging can render endothelial cells more susceptible to apoptosis through an oxidative stress-dependent pathway. We examined whether senescence-associated to oxidative stress determines the injury induced by the uremia in endothelial cells. Human umbilical vein endothelial cells (HUVEC) was incubated with human uremic serum and, in the animal model, endothelial cells were obtained from aortas of uremic and no uremic rats. Vitamin C was used to prevent oxidative stress. Senescence, assessed by telomere length and enzyme-betagalactosidase (β-gal), reactive oxygen species (ROS), mitochondrial depolarization (JC-1 probe), caspase 3, and apoptosis were determined by flow cytometry. NF-κB activity was determined by Western blot. Uremic serum increased ROS and NF-κB in young and aging HUVEC. However only in aging cells, uremic serum induced apoptosis (vs young HUVEC, p

Lack of Klotho expression in mice leads to premature aging and age-related diseases, including vascular diseases. The aim of this study was to determine how endothelial cell line senescence affects Klotho expression and whether intra- or extracellular Klotho has any effect on the response of senescent cells to oxidative stress. The study was performed using human endothelial cells (HUVEC); cell aging was obtained by prolongation of cell division to 42 population doublings (PD). Senescence was also obtained by exposure to TNFα, which causes cell changes resembling cellular senescence. The decline in Klotho preceded the manifestations of cell ageing: telomere shortening and β-galactosidase expression. Klotho was also reduced in cells exposed to the proinflammatory cytokine TNFα. The addition of exogenous Klotho to aging cells did not modify the proportion of cells with short telomeres or any other feature of cell aging; however, exogenous Klotho prevented the changes resembling premature cellular senescence associated with TNFα, such as the decrease in telomere length and the increase in β-galactosidase-positive cells. Likewise exogenous Klotho prevented the increases in reactive oxygen species (ROS) activity, mitochondrial potential and cell apoptosis induced by TNFα.

Cardiovascular complications are a major cause of mortality in hemodialysis patients. On-line hemofiltration combines convective clearance for removing large solutes with diffusion to remove small solutes and is associated with a significant reduction of inflammation and improved patient survival. We compared on-line hemofiltration to high-flux hemodialysis (HF-HD) in patients in a sequential manner. At baseline, 15 stable patients on HF-HD as compared with five control subjects showed significant increases in CD14+CD16+ cells, endothelial microparticles, and endothelial progenitor cells (EPCs). After 4 months of on-line hemofiltration, the number of CD14+CD16+ cells, microparticles, and EPCs decreased. After returning to HF-HD for 4 months, all measured parameters returned to their respective baseline values. The number of CD14+CD16+ cells correlated with both endothelial microparticles and EPCs. We conclude that on-line hemofiltration attenuates endothelial dysfunction possibly by decreasing microinflammation. This effect may be directly caused by a modulatory effect of on-line hemofiltration on proinflammatory cells or by a complex interaction that encompasses a wider removal of uremic toxins.

Background: Repeatedly stimulated mononuclear cells may become senescent prematurely as a result of frequent activation-induced replication. In hemodialysis patients, mononuclear cells are activated repeatedly with each hemodialysis procedure. Characteristics of senescent mononuclear cells include telomere length shortening, increased p53 expression, CD14dim/CD16bright expression, and interleukin overproduction. Methods: Peripheral mononuclear cells from 15 hemodialysis patients and 15 age-matched controls were evaluated. Telomere length was assessed by means of fluorescence in situ hybridization in flow cytometry. Expression of p53, CD14/CD16, and intracellular cytokine production (interleukin-1β [IL-1β], IL-6, and IL-4) was evaluated by means of flow cytometry using specific antibodies. Results: Features of senescence were found in a subpopulation of mononuclear cells: (1) accelerated telomere shortening, (2) increased p53 expression, (3) CD14dim/CD16bright expression, and (4) cytokine overproduction (IL-1β, IL-6, and IL-4). Telomere length shortening was present in 40% ± 6% of cells from hemodialysis patients compared with less than 5% from age-matched controls. Percentage of cells with short telomeres correlated positively with serum C-reactive protein level, which reflects inflammation. p53 expression was increased in mononuclear cells from hemodialysis patients. Mononuclear cells from hemodialysis patients with decreased telomere length mainly showed the CD14dim/CD16bright phenotype; conversely, cells with normal telomeres presented the CD14bright/CD16dim phenotype. Finally, mononuclear cells from hemodialysis patients, but not controls, spontaneously produced the proinflammatory cytokines IL-1β and IL-6. Conclusion: This study shows the presence of a prematurely senescent subpopulation of peripheral mononuclear cells in hemodialysis patients. These senescent cells probably result from repeated activation and may have a pathophysiological role in the chronic inflammation described in hemodialysis patients.

Repeated stimulation of immune cells may induce an “activation-induced cell death” (AICD) program. Allergy is characterized by the cyclic activation of allergen-reactive immune cells. To study the effects of allergen stimulation in cell proliferation and apoptosis in atopic subjects, peripheral blood mononuclear cells (PBL) from 40 atopic patients with positive reactivity to the allergens Olea Europaea (OE) and Lollium Perenne (LP) (20 without immunotherapy and 20 with specific immunotherapy) and 10 normal subjects were cultured with the allergens OE and LP. PBL from atopic patients proliferate more vigorously than cells from normal subjects after culture in vitro with both allergens, although PBL from atopic subjects without immunotherapy proliferate more than PBL from atopic subjects with immunotherapy. The study of cell proliferation shows that in atopic patients PBL mainly exhibit the CD4/CD45RO phenotype. This preferential proliferation is more evident in PBL from atopic patients treated without immunotherapy. Cell culture with specific allergens induces apoptosis in PBL from atopic patients. The percentage of apoptosis increased when atopic patients had been previously treated with immunotherapy. In addition to the observed increase in cell proliferation, apoptosis mainly occurs in the CD45RO cells that support the involvement of these cells in allergy. Furthermore, results obtained in cells from immunized patients suggest that an AICD process may partly at least explain the mechanism of action of allergen immunotherapy.

Immunosenescence is a process that primarily affects the T cell compartment of the immune system, although age-associated immunological alterations have also been demonstrated in the NK cell phenotype and function. A significant expansion in the number of NK cells is found in aging. The NK cytotoxic capacity of total peripheral blood lymphocytes is also well preserved, not only in healthy elderly people but also in centenarians. However, NK cell killing of K562 is impaired when considered in a per-cell basis, and this defect is associated with defective signal transduction after activation more than a diminished conjugate formation or killing capacity. We have studied the phenotype of NK cells in elderly donors fulfilling the Senieur criteria. We have also studied the capacity of these cells to be activated by IL2 when different NK cell functions, other than cytotoxicity, are considered. Our results confirm the increased percentage of NK cells in the elderly due to the expansion of the CD56dim subset that also show an altered pattern of activation markers, whereas no differences were found in the CD56bright subset. The response of NK cells to IL2 was found to be impaired when proliferation, expression of CD69, and Ca2+ mobilization were considered, whereas TNF-alpha production was not significantly affected. These results suggest that human NK cells do not escape the aging process, although senescence have a differential effect on distinct NK cell biological functions, ranging from severe to negligible impairment, depending on the parameters considered.