Proteomic analysis of endothelial cells and extracellular vesicles in response to indoxyl sulfate: Mechanisms of endothelial dysfunction in chronic kidney disease.

AIMS: Cardiovascular pathology is the main cause of death in chronic kidney disease (CKD) patients. CKD is associated with the accumulation of uremic toxins in the bloodstream, and indoxyl sulfate (IS) is one of the most abundant uremic toxins found in the blood of CKD patients. We conducted an in vitro study to assess the mechanisms underlying the IS-induced endothelial dysfunction that could lead to cardiovascular diseases. We also studied their extracellular vesicles (EVs) owing to their capacity to act as messengers that transmit signals through their cargo. MAIN METHODS: EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and tetraspanin expression. Cell lysates and isolated EVs were analyzed using liquid chromatography coupled with mass spectrometry, followed by Gene Set Enrichment Analysis to identify the altered pathways. KEY FINDINGS: Proteomic analysis of endothelial cells revealed that IS causes an increase in proteins related to adipogenesis, inflammation, and xenobiotic metabolism and a decrease in proliferation. Extracellular matrix elements, as well as proteins associated with myogenesis, response to UV irradiation, and inflammation, were found to be downregulated in IS-treated EVs. Fatty acid metabolism was also found to be increased along with adipogenesis and inflammation observed in cells. SIGNIFICANCE: The treatment of endothelial cells with IS increased the expression of proteins related to adipogenesis, inflammation, and xenobiotic metabolism and was less associated with proliferation. Furthermore, EVs from cells treated with IS may mediate endothelial dysfunction, since they present fewer extracellular matrix elements, myogenesis, inflammatory factors, and proteins downregulated in response to UV radiation.

Strategies for measuring non-evoked pain in preclinical models of neuropathic pain: Systematic review.

The development of new analgesics for neuropathic pain treatment is crucial. The failure of promising drugs in clinical trials may be related to the over-reliance on reflex-based responses (evoked pain) in preclinical drug testing, which may not fully represent clinical neuropathic pain, characterized by spontaneous non-evoked pain (NEP). Hence, strategies for assessing NEP in preclinical studies emerged. This systematic review identified 443 articles evaluating NEP in neuropathic pain models (mainly traumatic nerve injuries in male rodents). An exponential growth in NEP evaluation was observed, which was assessed using 48 different tests classified in 12 NEP-related outcomes: anxiety, exploration/locomotion, paw lifting, depression, conditioned place preference, gait, autotomy, wellbeing, facial grooming, cognitive impairment, facial pain expressions and vocalizations. Although most of these outcomes showed clear limitations, our analysis suggests that conditioning-associated outcomes, pain-related comorbidities, and gait evaluation may be the most effective strategies. Moreover, a minimal part of the studies evaluated standard analgesics. The greater emphasis on evaluating NEP aligning with clinical pain symptoms may enhance analgesic drug development, improving clinical translation.

Plasma glycocalyx pattern: a mirror of endothelial damage in chronic kidney disease.

Background: Endothelial damage and cardiovascular disease complicate chronic kidney disease (CKD). The increased atherogenicity observed in patients with CKD can be linked to microinflammation and endothelial damage. Circulating endothelial glycocalyx degradation products, such as perlecan and decorin, tend to be elevated in CKD. We aimed to explore the association between the plasma perlecan and decorin levels and this pro-inflammatory and atherogenic state by studying monocyte subpopulations and intracellular adhesion molecule (ICAM)-1 expression in patients with CKD. Methods: We studied 17 healthy controls, 23 patients with advanced CKD, 25 patients on haemodialysis, 23 patients on peritoneal dialysis and 20 patients who underwent kidney transplantation. Perlecan and decorin levels were evaluated using enzyme-linked immunosorbent assays, and the monocyte phenotype was analysed using direct immunofluorescence and flow cytometry. Results: The plasma perlecan levels were higher in patients with CKD than in the healthy controls. These levels were associated with a higher prevalence of ICAM-1+ monocytes. Conversely, patients with advanced CKD (pre-dialysis) had higher plasma decorin levels, which were associated with a reduced ICAM-1 expression per monocyte. Conclusions: Elevated perlecan levels in CKD may be associated with a higher prevalence of ICAM-1+ monocytes and a pro-inflammatory phenotype. Elevated decorin levels may act as a negative regulator of ICAM-1 expression in monocytes. Therefore, perlecan and decorin may be related to inflammation and monocyte activation in CKD and may act as potential markers of endothelial damage.

New mechanisms involved in the development of cardiovascular disease in chronic kidney disease.

Chronic kidney disease (CKD) is a pathology with a high worldwide incidence and an upward trend affecting the elderly. When CKD is very advanced, the use of renal replacement therapies is required to prolong its life (dialysis or kidney transplantation). Although dialysis improves many complications of CKD, the disease does not reverse completely. These patients present an increase in oxidative stress, chronic inflammation and the release of extracellular vesicles (EVs), which cause endothelial damage and the development of different cardiovascular diseases (CVD). CKD patients develop premature diseases associated with advanced age, such as CVD. EVs play an essential role in developing CVD in patients with CKD since their number increases in plasma and their content is modified. The EVs of patients with CKD cause endothelial dysfunction, senescence and vascular calcification. In addition, miRNAs free or transported in EVs together with other components carried in these EVs promote endothelial dysfunction, thrombotic and vascular calcification in CKD, among other effects. This review describes the classic factors and focuses on the role of new mechanisms involved in the development of CVD associated with CKD, emphasizing the role of EVs in the development of cardiovascular pathologies in the context of CKD. Moreover, the review summarized the EVs' role as diagnostic and therapeutic tools, acting on EV release or content to avoid the development of CVD in CKD patients.

Vitamin D, Cellular Senescence and Chronic Kidney Diseases: What Is Missing in the Equation?

As life expectancy increases in many countries, the prevalence of age-related diseases also rises. Among these conditions, chronic kidney disease is predicted to become the second cause of death in some countries before the end of the century. An important problem with kidney diseases is the lack of biomarkers to detect early damage or to predict the progression to renal failure. In addition, current treatments only retard kidney disease progression, and better tools are needed. Preclinical research has shown the involvement of the activation of cellular senescence-related mechanisms in natural aging and kidney injury. Intensive research is searching for novel treatments for kidney diseases as well as for anti-aging therapies. In this sense, many experimental shreds of evidence support that treatment with vitamin D or its analogs can exert pleiotropic protective effects in kidney injury. Moreover, vitamin D deficiency has been described in patients with kidney diseases. Here, we review recent evidence about the relationship between vitamin D and kidney diseases, explaining the underlying mechanisms of the effect of vitamin D actions, with particular attention to the modulation of cellular senescence mechanisms.

Extracellular Vesicles as "Very Important Particles" (VIPs) in Aging.

In recent decades, extracellular vesicles have been recognized as "very important particles" (VIPs) associated with aging and age-related disease. During the 1980s, researchers discovered that these vesicle particles released by cells were not debris but signaling molecules carrying cargoes that play key roles in physiological processes and physiopathological modulation. Following the International Society for Extracellular Vesicles (ISEV) recommendation, different vesicle particles (e.g., exosomes, microvesicles, oncosomes) have been named globally extracellular vesicles. These vesicles are essential to maintain body homeostasis owing to their essential and evolutionarily conserved role in cellular communication and interaction with different tissues. Furthermore, recent studies have shown the role of extracellular vesicles in aging and age-associated diseases. This review summarizes the advances in the study of extracellular vesicles, mainly focusing on recently refined methods for their isolation and characterization. In addition, the role of extracellular vesicles in cell signaling and maintenance of homeostasis, as well as their usefulness as new biomarkers and therapeutic agents in aging and age-associated diseases, has also been highlighted.

Particulate Matter in Human Elderly: Higher Susceptibility to Cognitive Decline and Age-Related Diseases.

This review highlights the significant impact of air quality, specifically particulate matter (PM), on cognitive decline and age-related diseases in the elderly. Despite established links to other pathologies, such as respiratory and cardiovascular illnesses, there is a pressing need for increased attention to the association between air pollution and cognitive aging, given the rising prevalence of neurocognitive disorders. PM sources are from diverse origins, including industrial activities and combustion engines, categorized into PM10, PM2.5, and ultrafine PM (UFPM), and emphasized health risks from both outdoor and indoor exposure. Long-term PM exposure, notably PM2.5, has correlated with declines in cognitive function, with a specific vulnerability observed in women. Recently, extracellular vesicles (EVs) have been explored due to the interplay between them, PM exposure, and human aging, highlighting the crucial role of EVs, especially exosomes, in mediating the complex relationship between PM exposure and chronic diseases, particularly neurological disorders. To sum up, we have compiled the pieces of evidence that show the potential contribution of PM exposure to cognitive aging and the role of EVs in mediating PM-induced cognitive impairment, which presents a promising avenue for future research and development of therapeutic strategies. Finally, this review emphasizes the need for policy changes and increased public awareness to mitigate air pollution, especially among vulnerable populations such as the elderly.

The Contribution of Extracellular Vesicles From Senescent Endothelial and Vascular Smooth Muscle Cells to Vascular Calcification.

Vascular calcification is an irreversible pathological process associated with a loss of vascular wall function. This process occurs as a result of aging and age-related diseases, such as cardiovascular and chronic kidney diseases, and leads to comorbidities. During these age-related diseases, the endothelium accumulates senescent cells, which stimulate calcification in vascular smooth muscle cells. Currently, vascular calcification is a silent pathology, and there are no early diagnostic tools. Therefore, by the time vascular calcification is diagnosed, it is usually untreatable. Some mediators, such as oxidative stress, inflammation, and extracellular vesicles, are inducers and promoters of vascular calcification. They play a crucial role during vascular generation and the progression of vascular calcification. Extracellular vesicles, mainly derived from injured endothelial cells that have acquired a senescent phenotype, contribute to calcification in a manner mostly dependent on two factors: (1) the number of extracellular vesicles released, and (2) their cargo. In this review, we present state-of-the-art knowledge on the composition and functions of extracellular vesicles involved in the generation and progression of vascular calcification.

Special Issue "Oxidative Stress in Aging and Associated Chronic Diseases".

Aging is a risk factor for several diseases, including cardiovascular disease, type 2 diabetes, hypertension, cancer, osteoarthritis, and Alzheimer; oxidative stress is a key player in the development and progression of aging and age-associated diseases [...].

Endothelial Senescence and the Chronic Vascular Diseases: Challenges and Therapeutic Opportunities in Atherosclerosis.

Atherosclerosis is probably one of the paradigms of disease linked to aging. Underlying the physiopathology of atherosclerosis are cellular senescence, oxidative stress, and inflammation. These factors are increased in the elderly and from chronic disease patients. Elevated levels of oxidative stress affect cellular function and metabolism, inducing senescence. This senescence modifies the cell phenotype into a senescent secretory phenotype. This phenotype activates immune cells, leading to chronic systemic inflammation. Moreover, due to their secretory phenotype, senescence cells present an increased release of highlighted extracellular vesicles that will change nearby/neighborhood cells and paracrine signaling. For this reason, searching for specific senescent cell biomarkers and therapies against the development/killing of senescent cells has become relevant. Recently, senomorphic and senolityc drugs have become relevant in slowing down or eliminating senescence cells. However, even though they have shown promising results in experimental studies, their clinical use is still yet to be determined.

Effect of Kidney Transplantation on Accelerated Immunosenescence and Vascular Changes Induced by Chronic Kidney Disease.

Kidney transplantation is the best option for patients with end-stage renal disease. Despite the improvement in cardiovascular burden (leading cause of mortality among patients with chronic kidney disease), cardiovascular adverse outcomes related to the inflammatory process remain a problem. Thus, the aim of the present study was to characterize the immune profile and microvesicles of patients who underwent transplantation. We investigated the lymphocyte phenotype (CD3, CD4, CD8, CD19, and CD56) and monocyte phenotype (CD14, CD16, CD86, and CD54) in peripheral blood, and endothelium-derived microvesicles (annexin V+CD31+CD41-) in plasma of patients with advanced chronic kidney disease (n = 40), patients with transplantation (n = 40), and healthy subjects (n = 18) recruited from the University Hospital "12 de Octubre" (Madrid, Spain). Patients with kidney transplantation had B-cell lymphopenia, an impairment in co-stimulatory (CD86) and adhesion (CD54) molecules in monocytes, and a reduction in endothelium-derived microvesicles in plasma. The correlations between those parameters explained the modifications in the expression of co-stimulatory and adhesion molecules in monocytes caused by changes in lymphocyte populations, as well as the increase in the levels of endothelial-derived microvesicles in plasma caused by changes in lymphocyte and monocytes populations. Immunosuppressive treatment could directly or indirectly induce those changes. Nevertheless, the particular characteristics of these cells may partly explain the persistence of cardiovascular and renal alterations in patients who underwent transplantation, along with the decrease in arteriosclerotic events compared with advanced chronic kidney disease. In conclusion, the expression of adhesion molecules by monocytes and endothelial-derived microvesicles is related to lymphocyte alterations in patients with kidney transplantation.

Premature Aging in Chronic Kidney Disease: The Outcome of Persistent Inflammation beyond the Bounds.

Over the last hundred years, life expectancy in developed countries has increased because of healthier living habits and the treatment of chronic pathologies causing premature aging. Aging is an inexorable, time-dependent, multifactorial process characterized by a series of progressive and irreversible physiological changes associated with loss of functional, psychological, and social capabilities. Numerous factors, such as oxidative stress, inflammation, and cellular senescence, and an irreversible geriatric syndrome known as frailty, contribute to human body deterioration in aging. The speed of aging may differ between individuals depending on the presence or absence of multiple factors (genetic and/or environment) and the subsequent misbalance of homeostasis, together with the increase of frailty, which also plays a key role in developing chronic diseases. In addition, pathological circumstances have been reported to precipitate or accelerate the aging process. This review investigated the mechanisms involved in the developing pathologies, particularly chronic kidney disease, associated with aging.

A high magnesium concentration in citrate dialysate prevents oxidative stress and damage in human monocytes in vitro.

BACKGROUND: The use of dialysis fluids (DFs) during haemodialysis has been associated with increased oxidative stress and reduced serum magnesium (Mg) levels, contributing to chronic inflammation. Since the role of Mg in modulating immune function and reducing oxidative stress has been demonstrated, the aim of this study was to characterize in vitro whether increasing the Mg concentration in DFs could protect immune cells from oxidative stress and damage. METHODS: The effect of citrate [citrate dialysis fluid (CDF), 1 mM] or acetate [acetate dialysis fluid (ADF), 3 mM] dialysates with low (0.5 mM; routinely used) or high (1 mM, 1.25 mM and 2 mM) Mg concentrations was assessed in THP-1 human monocytes. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and oxidized/reduced (GSSG/GSH) glutathione were quantified under basal and inflammatory conditions (stimulation with lipopolysaccharide, LPS). RESULTS: The increase of Mg in CDF resulted in a significant reduction of ROS production under basal and inflammatory conditions (extremely marked in 2 mM Mg; P < 0.001). These effects were not observed in ADF. Interestingly, in a dose-dependent manner, high Mg doses in CDF reduced oxidative stress in monocytes under both basal and inflammatory conditions. In fact, 2 mM Mg significantly decreased the levels of GSH, GSSG and MDA and the GSSG/GSH ratio in relation to 0.5 mM Mg. CONCLUSIONS: CDF produces lower oxidative stress than ADF. The increase of Mg content in DFs, especially in CDF, could have a positive and protective effect in reducing oxidative stress and damage in immune cells, especially under inflammatory conditions.

Exploring New Kingdoms: The Role of Extracellular Vesicles in Oxi-Inflamm-Aging Related to Cardiorenal Syndrome.

The incidence of age associated chronic diseases has increased in recent years. Although several diverse causes produce these phenomena, abundant evidence shows that oxidative stress plays a central role. In recent years, numerous studies have focused on elucidating the role of oxidative stress in the development and progression of both aging and chronic diseases, opening the door to the discovery of new underlying mechanisms and signaling pathways. Among them, senolytics and senomorphics, and extracellular vesicles offer new therapeutic strategies to slow the development of aging and its associated chronic diseases by decreasing oxidative stress. In this review, we aim to discuss the role of extracellular vesicles in human cardiorenal syndrome development and their possible role as biomarkers, targets, or vehicles of drugs to treat this syndrome.

Microvesicles from indoxyl sulfate-treated endothelial cells induce vascular calcification in vitro.

Vascular calcification (VC), an unpredictable pathophysiological process and critical event in patients with cardiovascular diseases (CVDs), is the leading cause of morbi-mortality and disability in chronic kidney disease (CKD) patients worldwide. Currently, no diagnostic method is available for identifying patients at risk of VC development; the pathology is detected when the process is irreversible. Extracellular vesicles (EVs) from endothelial cells might promote VC. Therefore, their evaluation and characterization could be useful for designing new diagnostic tools. The aim of the present study is to investigate whether microvesicles (MVs) from endothelial cells damaged by uremic toxin and indoxyl sulfate (IS) could induce calcification in human vascular smooth muscle cells (VMSCs). Besides, we have also analyzed the molecular mechanisms by which these endothelial MVs can promote VC development. Endothelial damage has been evaluated according to the percentage of senescence in endothelial cells, differential microRNAs in endothelial cells, and the amount of MVs released per cell. To identify the role of MVs in VC, VSMCs were treated with MVs from IS-treated endothelial cells. Calcium, inflammatory gene expression, and procalcification mediator levels in VSMCs were determined. IS-treated endothelial cells underwent senescence and exhibited modulated microRNA expression and an increase in the release of MVs. VSMCs exposed to these MVs modulated the expression of pro-inflammatory genes and some mediators involved in calcification progression. MVs produced by IS-treated endothelial cells promoted calcification in VSMCs.

Increasing the Magnesium Concentration in Various Dialysate Solutions Differentially Modulates Oxidative Stress in a Human Monocyte Cell Line.

Oxidative stress is exacerbated in hemodialysis patients by several factors, including the uremic environment and the use of dialysis fluids (DFs). Since magnesium (Mg) plays a key role in modulating immune function and in reducing oxidative stress, we aimed to evaluate whether increasing the Mg concentration in different DFs could protect against oxidative stress in immunocompetent cells in vitro. Effect of ADF (acetate 3 mM), CDF (citrate 1 mM), and ACDF (citrate 0.8 mM + acetate 0.3 mM) dialysates with Mg at standard (0.5 mM) or higher (1, 1.25, and 2 mM) concentrations were assessed in THP-1 monocyte cultures. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were quantified under basal and uremic conditions (indoxyl sulfate (IS) treatment). Under uremic conditions, the three DFs with 0.5 mM Mg promoted higher ROS production and lipid damage than the control solution. However, CDF and ACDF induced lower levels of ROS and MDA, compared to that induced by ADF. High Mg concentration (1.25 and/or 2 mM) in CDF and ACDF protected against oxidative stress, indicated by reduced ROS and MDA levels compared to respective DFs with standard concentration of Mg. Increasing Mg concentrations in ADF promoted high ROS production and MDA content. Thus, an increase in Mg content in DFs has differential effects on the oxidative stress in IS-treated THP-1 cells depending on the dialysate used.

Mechanisms of Cardiovascular Disorders in Patients With Chronic Kidney Disease: A Process Related to Accelerated Senescence.

Cardiovascular diseases (CVDs), especially those involving a systemic inflammatory process such as atherosclerosis, remain the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). CKD is a systemic condition affecting approximately 10% of the general population. The prevalence of CKD has increased over the past decades because of the aging of the population worldwide. Indeed, CVDs in patients with CKD constitute a premature form of CVD observed in the general population. Multiple studies indicate that patients with renal disease undergo accelerated aging, which precipitates the appearance of pathologies, including CVDs, usually associated with advanced age. In this review, we discuss several aspects that characterize CKD-associated CVDs, such as etiopathogenic elements that CKD patients share with the general population, changes in the cellular balance of reactive oxygen species (ROS), and the associated process of cellular senescence. Uremia-associated aging is linked with numerous changes at the cellular and molecular level. These changes are similar to those observed in the normal process of physiologic aging. We also discuss new perspectives in the study of CKD-associated CVDs and epigenetic alterations in intercellular signaling, mediated by microRNAs and/or extracellular vesicles (EVs), which promote vascular damage and subsequent development of CVD. Understanding the processes and factors involved in accelerated senescence and other abnormal intercellular signaling will identify new therapeutic targets and lead to improved methods of diagnosis and monitoring for patients with CKD-associated CVDs.

Hypoxia-Inducible Factor-1α: The Master Regulator of Endothelial Cell Senescence in Vascular Aging.

Aging is one of the hottest topics in biomedical research. Advances in research and medicine have helped to preserve human health, leading to an extension of life expectancy. However, the extension of life is an irreversible process that is accompanied by the development of aging-related conditions such as weakness, slower metabolism, and stiffness of vessels. It also debated that aging can be considered an actual disease with aging-derived comorbidities, including cancer or cardiovascular disease. Currently, cardiovascular disorders, including atherosclerosis, are considered as premature aging and represent the first causes of death in developed countries, accounting for 31% of annual deaths globally. Emerging evidence has identified hypoxia-inducible factor-1α as a critical transcription factor with an essential role in aging-related pathology, in particular, regulating cellular senescence associated with cardiovascular aging. In this review, we will focus on the regulation of senescence mediated by hypoxia-inducible factor-1α in age-related pathologies, with particular emphasis on the crosstalk between endothelial and vascular cells in age-associated atherosclerotic lesions. More specifically, we will focus on the characteristics and mechanisms by which cells within the vascular wall, including endothelial and vascular cells, achieve a senescent phenotype.

MicroRNA-126 regulates Hypoxia-Inducible Factor-1α which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence.

Whereas a healthy endothelium maintains physiological vascular functions, endothelial damage contributes to the development of cardiovascular diseases. Endothelial senescence is the main determinant of endothelial dysfunction and thus of age-related cardiovascular disease. The objective of this study is to test the involvement of microRNA-126 and HIF-1α in a model of replicative endothelial senescence and the interrelationship between both molecules in this in vitro model. We demonstrated that senescent endothelial cells experience impaired tube formation and delayed wound healing. Senescent endothelial cells failed to express HIF-1α, and the microvesicles released by these cells failed to carry HIF-1α. Of note, HIF-1α protein levels were restored in HIF-1α stabilizer-treated senescent endothelial cells. Finally, we show that microRNA-126 was downregulated in senescent endothelial cells and microvesicles. With regard to the interplay between microRNA-126 and HIF-1α, transfection with a microRNA-126 inhibitor downregulated HIF-1α expression in early passage endothelial cells. Moreover, while HIF-1α inhibition reduced tube formation and wound healing closure, microRNA-126 levels remained unchanged. These data indicate that HIF-1α is a target of miRNA-126 in protective and reparative functions, and suggest that their therapeutic modulation could benefit age-related vascular disease.