Digital methylation-specific PCR: New applications for liquid biopsy.

Epigenetic analysis is a fundamental part of understanding pathophysiological processes with potential applications in diagnosis, prognosis, and assessment of disease susceptibility. Epigenetic changes have been widely studied in chronic obstructive pulmonary disease (COPD), but currently, there is no molecular marker used to improve the treatment of patients. Furthermore, this progressive disease is a risk factor for the development of more severe COVID-19. Methylation-specific polymerase chain reaction (MSP-PCR) plays an important role in the analysis of DNA methylation profiles, and it is one of the most widely used techniques. In this context, the combination of MSP-PCR with emerging PCR technologies, such as digital PCR (dPCR), results in more accurate analyses of the DNA methylation profile of the genes under study. In this study, we propose the application of the MSP-dPCR technique to evaluate the methylation profile of the ADAM33 gene from saliva samples and lung tissue biopsies of patients with COPD and COVID-19. MSP-dPCR generated a measurable prediction of gene methylation rate, with the potential application of this combined technology for diagnostic and prognostic purposes. It has also proven to be a powerful tool for liquid biopsy applications.

The Role of α3ß1 Integrin Modulation on Fabry Disease Podocyte Injury and Kidney Impairment.

Podocyte dysfunction plays a crucial role in renal injury and is identified as a key contributor to proteinuria in Fabry disease (FD), primarily impacting glomerular filtration function (GFF). The α3ß1 integrins are important for podocyte adhesion to the glomerular basement membrane, and disturbances in these integrins can lead to podocyte injury. Therefore, this study aimed to assess the effects of chloroquine (CQ) on podocytes, as this drug can be used to obtain an in vitro condition analogous to the FD. Murine podocytes were employed in our experiments. The results revealed a dose-dependent reduction in cell viability. CQ at a sub-lethal concentration (1.0 µg/mL) induced lysosomal accumulation significantly (p < 0.0001). Morphological changes were evident through scanning electron microscopy and immunofluorescence, highlighting alterations in F-actin and nucleus morphology. No significant changes were observed in the gene expression of α3ß1 integrins via RT-qPCR. Protein expression of α3 integrin was evaluated with Western Blotting and immunofluorescence, demonstrating its lower detection in podocytes exposed to CQ. Our findings propose a novel in vitro model for exploring secondary Fabry nephropathy, indicating a modulation of α3ß1 integrin and morphological alterations in podocytes under the influence of CQ.

The Interplay between Uremic Toxins and Albumin, Membrane Transporters and Drug Interaction.

Uremic toxins are a heterogeneous group of molecules that accumulate in the body due to the progression of chronic kidney disease (CKD). These toxins are associated with kidney dysfunction and the development of comorbidities in patients with CKD, being only partially eliminated by dialysis therapies. Importantly, drugs used in clinical treatments may affect the levels of uremic toxins, their tissue disposition, and even their elimination through the interaction of both with proteins such as albumin and cell membrane transporters. In this context, protein-bound uremic toxins (PBUTs) are highlighted for their high affinity for albumin, the most abundant serum protein with multiple binding sites and an ability to interact with drugs. Membrane transporters mediate the cellular influx and efflux of various uremic toxins, which may also compete with drugs as substrates, and both may alter transporter activity or expression. Therefore, this review explores the interaction mechanisms between uremic toxins and albumin, as well as membrane transporters, considering their potential relationship with drugs used in clinical practice.

Extracellular Vesicles and Their Relationship with the Heart-Kidney Axis, Uremia and Peritoneal Dialysis.

Cardiorenal syndrome (CRS) is described as primary dysfunction in the heart culminating in renal injury or vice versa. CRS can be classified into five groups, and uremic toxin (UT) accumulation is observed in all types of CRS. Protein-bound uremic toxin (PBUT) accumulation is responsible for permanent damage to the renal tissue, and mainly occurs in CRS types 3 and 4, thus compromising renal function directly leading to a reduction in the glomerular filtration rate (GFR) and/or subsequent proteinuria. With this decrease in GFR, patients may need renal replacement therapy (RRT), such as peritoneal dialysis (PD). PD is a high-quality and home-based dialysis therapy for patients with end-stage renal disease (ESRD) and is based on the semi-permeable characteristics of the peritoneum. These patients are exposed to factors which may cause several modifications on the peritoneal membrane. The presence of UT may harm the peritoneum membrane, which in turn can lead to the formation of extracellular vesicles (EVs). EVs are released by almost all cell types and contain lipids, nucleic acids, metabolites, membrane proteins, and cytosolic components from their cell origin. Our research group previously demonstrated that the EVs can be related to endothelial dysfunction and are formed when UTs are in contact with the endothelial monolayer. In this scenario, this review explores the mechanisms of EV formation in CRS, uremia, the peritoneum, and as potential biomarkers in peritoneal dialysis.

Heat Shock Proteins: Connectors between Heart and Kidney.

Over the development of eukaryotic cells, intrinsic mechanisms have been developed in order to provide the ability to defend against aggressive agents. In this sense, a group of proteins plays a crucial role in controlling the production of several proteins, guaranteeing cell survival. The heat shock proteins (HSPs), are a family of proteins that have been linked to different cellular functions, being activated under conditions of cellular stress, not only imposed by thermal variation but also toxins, radiation, infectious agents, hypoxia, etc. Regarding pathological situations as seen in cardiorenal syndrome (CRS), HSPs have been shown to be important mediators involved in the control of gene transcription and intracellular signaling, in addition to be an important connector with the immune system. CRS is classified as acute or chronic and according to the first organ to suffer the injury, which can be the heart (CRS type 1 and type 2), kidneys (CRS type 3 and 4) or both (CRS type 5). In all types of CRS, the immune system, redox balance, mitochondrial dysfunction, and tissue remodeling have been the subject of numerous studies in the literature in order to elucidate mechanisms and propose new therapeutic strategies. In this sense, HSPs have been targeted by researchers as important connectors between kidney and heart. Thus, the present review has a focus to present the state of the art regarding the role of HSPs in the pathophysiology of cardiac and renal alterations, as well their role in the kidney-heart axis.

Uremic Toxins: An Alarming Danger Concerning the Cardiovascular System.

The kidneys and heart share functions with the common goal of maintaining homeostasis. When kidney injury occurs, many compounds, the so-called "uremic retention solutes" or "uremic toxins," accumulate in the circulation targeting other tissues. The accumulation of uremic toxins such as p-cresyl sulfate, indoxyl sulfate and inorganic phosphate leads to a loss of a substantial number of body functions. Although the concept of uremic toxins is dated to the 1960s, the molecular mechanisms capable of leading to renal and cardiovascular injuries are not yet known. Besides, the greatest toxic effects appear to be induced by compounds that are difficult to remove by dialysis. Considering the close relationship between renal and cardiovascular functions, an understanding of the mechanisms involved in the production, clearance and overall impact of uremic toxins is extremely relevant for the understanding of pathologies of the cardiovascular system. Thus, the present study has as main focus to present an extensive review on the impact of uremic toxins in the cardiovascular system, bringing the state of the art on the subject as well as clinical implications related to patient's therapy affected by chronic kidney disease, which represents high mortality of patients with cardiac comorbidities.

Uremic endothelial-derived extracellular vesicles: Mechanisms of formation and their role in cell adhesion, cell migration, inflammation, and oxidative stress.

p-Cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) are uremic toxins found in chronic kidney disease (CKD) that are closely related to endothelial extracellular vesicles (EVs) formation. The present study aimed to understand the role of EVs and their role in cell adhesion and migration, inflammation, and oxidative stress. Human endothelial cells were treated with PCS, IS, and Pi in pre-established uremic and kinetic recommendations. EVs were characterized using scanning electron microscopy, flow cytometry, and NanoSight assays. The concentrations of EVs were established using Alamar Blue and MTT assays. Cell adhesion to extracellular matrix proteins was analyzed using an adhesion assay. Inflammation and oxidative stress were assessed by vascular cell adhesion molecule-1 expression/monocyte migration and reactive oxygen species production, respectively. The capacity of EVs to stimulate endothelial cell migration was evaluated using a wound-healing assay. Our data showed that endothelial cells stimulated with uremic toxins can induce the formation of EVs of different sizes, quantities, and concentrations, depending on the uremic toxin used. Cell adhesion was significantly (P < 0.01) stimulated in cells exposed to PCS-induced extracellular vesicles (PCSEVs) and inorganic phosphate-induced extracellular vesicles (PiEVs). Cell migration was significantly (P < 0.05) stimulated by PCSEVs. VCAM-1 expression was evident in cells treated with PCSEVs and IS-induced extracellular vesicles (ISEVs). EVs are not able to stimulate monocyte migration or oxidative stress. In conclusion, EVs may be a biomarker of endothelial injury and the inflammatory process, playing an important role in cell-to-cell communication and pathophysiological processes, although more studies are needed to better understand the mechanisms of EVs in uremia.

How do Uremic Toxins Affect the Endothelium?

Uremic toxins can induce endothelial dysfunction in patients with chronic kidney disease (CKD). Indeed, the structure of the endothelial monolayer is damaged in CKD, and studies have shown that the uremic toxins contribute to the loss of cell-cell junctions, increasing permeability. Membrane proteins, such as transporters and receptors, can mediate the interaction between uremic toxins and endothelial cells. In these cells, uremic toxins induce oxidative stress and activation of signaling pathways, including the aryl hydrocarbon receptor (AhR), nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) pathways. The activation of these pathways leads to overexpression of proinflammatory (e.g., monocyte chemoattractant protein-1, E-selectin) and prothrombotic (e.g., tissue factor) proteins. Uremic toxins also induce the formation of endothelial microparticles (EMPs), which can lead to the activation and dysfunction of other cells, and modulate the expression of microRNAs that have an important role in the regulation of cellular processes. The resulting endothelial dysfunction contributes to the pathogenesis of cardiovascular diseases, such as atherosclerosis and thrombotic events. Therefore, uremic toxins as well as the pathways they modulated may be potential targets for therapies in order to improve treatment for patients with CKD.

Indoxyl Sulfate Contributes to Uremic Sarcopenia by Inducing Apoptosis in Myoblasts.

OBJETIVE: Uremic sarcopenia is a complication of chronic kidney disease, particularly in its later stages, which leads to musculoskeletal disability. Uremic toxins have been linked to the pathogenesis of several manifestations of uremic syndrome. We sought to investigate whether indoxyl sulphate (IS), a protein-bound uremic toxin, is implicated in the development of uremic sarcopenia. MATERIAL AND METHODS: Myoblasts were exposed to IS at normal (0.6 mg/L, IS0.6), uremic (53 mg/L, IS53) or maximum uremic (236 mg/L, IS236) concentrations for 24, 48 and 72 h. Cell viability was evaluated by MTT assay and by 7-aminoactinomycin D staining. ROS generation and apoptosis were evaluated by flow cytometry. MyoD and myogenin mRNA expression was evaluated by qRT-PCR and myosin heavy chain expression by immunocytochemistry. RESULTS: Myoblast viability was reduced by IS236 in a time-dependent pattern (p <0.05; 84.4, 68.0, and 63.6%). ROS production was significantly higher (p <0.05) in cells exposed to IS53 and IS236 compared to control (untreated cells). The apoptosis rate was significantly higher in cells treated with IS53 and IS236 than in control after 48h (p <0.05; 4.7 ± 0.1% and 4.6 ± 0.3% vs. 3.1 ± 0.1%, respectively) and 72h (p <0.05; 9.6 ± 1.1% and 10.4 ± 0.3% vs. 3.1 ± 0.7%, respectively). No effect was observed on MyoD, myogenin, myosin heavy chain expression, and markers of myoblast differentiation at any IS concentration tested or time-point experiment. CONCLUSIONS: These data indicate that IS has direct toxic effects on myoblast by decreasing its viability and increasing cell apoptosis. IS may be a potential target for treating uremic sarcopenia.

Endothelial Microparticles in Uremia: Biomarkers and Potential Therapeutic Targets.

Endothelial microparticles (EMPs) are vesicles derived from cell membranes, which contain outsourced phosphatidylserine and express adhesion molecules, such as cadherin, intercellular cell adhesion molecule-1 (ICAM-1), E-selectin, and integrins. EMPs are expressed under physiological conditions and continue circulating in the plasma. However, in pathologic conditions their levels increase, and they assume a pro-inflammatory and pro-coagulant role via interactions with monocytes; these effects are related to the development of atherosclerosis. Chronic kidney dysfunction (CKD) characterizes this dysfunctional scenario through the accumulation of uremic solutes in the circulating plasma, whose toxicity is related to the development of cardiovascular diseases. Therefore, this review aims to discuss the formation of EMPs and their biological effects in the uremic environment. Data from previous research demonstrate that uremic toxins are closely associated with the activation of inflammatory biomarkers, cardiovascular dysfunction processes, and the release of EMPs. The impact of a decrease in circulating EMPs in clinical studies has not yet been evaluated. Thus, whether MPs are biochemical markers and/or therapeutic targets has yet to be established.