Impact of Uremic Toxins on Endothelial Dysfunction in Chronic Kidney Disease: A Systematic Review

Patients with chronic kidney disease (CKD) are at a highly increased risk of cardiovascular complications, with increased vascular inflammation, accelerated atherogenesis and enhanced thrombotic risk. Considering the central role of the endothelium in protecting from atherogenesis and thrombosis, as well as its cardioprotective role in regulating vasorelaxation, this study aimed to systematically integrate literature on CKD-associated endothelial dysfunction, including the underlying molecular mechanisms, into a comprehensive overview. Therefore, we conducted a systematic review of literature describing uremic serum or uremic toxin-induced vascular dysfunction with a special focus on the endothelium. This revealed 39 studies analyzing the effects of uremic serum or the uremic toxins indoxyl sulfate, cyanate, modified LDL, the advanced glycation end products N-carboxymethyl-lysine and N-carboxyethyl-lysine, p-cresol and p-cresyl sulfate, phosphate, uric acid and asymmetric dimethylarginine. Most studies described an increase in inflammation, oxidative stress, leukocyte migration and adhesion, cell death and a thrombotic phenotype upon uremic conditions or uremic toxin treatment of endothelial cells. Cellular signaling pathways that were frequently activated included the ROS, MAPK/NF-κB, the Aryl-Hydrocarbon-Receptor and RAGE pathways. Overall, this review provides detailed insights into pathophysiological and molecular mechanisms underlying endothelial dysfunction in CKD. Targeting these pathways may provide new therapeutic strategies reducing increased the cardiovascular risk in CKD.

Uremic serum damages endothelium by provoking excessive neutrophil extracellular trap formation

Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, HV-derived neutrophils and differentiated HL-60 cells by retinoic acid were used to determine the effect of uremic serum-induced NETs on human umbilical vein EC (HUVEC). The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality, or vascular access failure. In vitro, HD-derived uremic serum significantly increased NET formation both in dHL-60 and isolated neutrophils compared to control serum, and these NETs decreased EC viability and induced their apoptosis. In addition, the level of ICAM-1, E-selectin and von Willebrand factor in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction.

Gene Expression Profile of HCAEC Cells Exposed to Serum From Chronic Kidney Disease Patients: Role of MAPK Signaling Pathway

End-stage renal disease (ESRD) patients have an elevated risk of cardiovascular (CV) complications including acute myocardial infarction (AMI); endothelial dysfunction and accumulation of uremic toxins have been associated with such CV-events. To explore which molecular pathways are involved in this CV-complication and the effects of the uremic serum on gene expression, an endothelial dysfunction model was studied through microarrays and pathway analysis. mRNA was isolated of human coronary arterial endothelial cells (HCAEC) primary cultures supplemented with 20% uremic serum from two groups of patients, USI: ESRD-patients; UCI: ESRD-AMI-patients. Affymetrix GeneChip® microarray and the LIMMA-package (Linear Models for Microarray Data) of the Bioconductor sofware17 was implemented to identify relevant DEGs between the two groups of uremic patients. Protein-protein interaction networks and pathway analysis were made to analyze the interaction and expression tendency of differentially expressed genes. 100 differentially expressed genes were identified from two data sets triggered by uremic state using bioinformatics, from 16,607. After in a new cohort, 30 genes were overexpressed in UCI group, which we identified 500 ontological genetic terms and one KEGG-pathway with p < 0.05. The metabolic pathway significantly represented was the MAPK signaling pathway. Network analysis showed six genes (PTGS2, SELE, ICAM1, HMOX1, EGR1, and TLR2) that represent potential markers for ESRD with AMI, as an approximation to their underlying mechanisms. The results obtained suggest that uremic toxins in patients with ESRD can alter HCAEC and modify the gene expression profile, which could have an impact on the development of cardiovascular complications in these patients.

Uremia and Inadequate Oxygen Supply Induce Eryptosis and Intracellular Hypoxia in Red Blood Cells

BACKGROUND/AIMS: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition. METHODS: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography. RESULTS: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis. CONCLUSION: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation.

Uremic Serum Damages Endothelium by Provoking Excessive Neutrophil Extracellular Trap Formation

Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, we differentiated HL-60 cells into neutrophil-like cells (dHL-60) by applying retinoic acid and determined the effect of uremic serum on these dHL-60 and human umbilical vein EC (HUVEC). The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality or vascular access failure. In vitro, HD-derived serum significantly increased NET formation by dHL-60, and these NETs decreased EC viability and induced their apoptosis. In addition, the ICAM-1 level in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction.

Macrophage and adipocyte interaction as a source of inflammation in kidney disease

In obesity, adipose tissue derived inflammation is associated with unfavorable metabolic consequences. Uremic inflammation is prevalent and contributes to detrimental outcomes. However, the contribution of adipose tissue inflammation in uremia has not been characterized. We studied the contribution of adipose tissue to uremic inflammation in-vitro, in-vivo and in human samples. Exposure to uremic serum resulted in activation of inflammatory pathways including NFκB and HIF1, upregulation of inflammatory cytokines/chemokines and catabolism with lipolysis, and lactate production. Also, co-culture of adipocytes with macrophages primed by uremic serum resulted in higher inflammatory cytokine expression than adipocytes exposed only to uremic serum. Adipose tissue of end stage renal disease subjects revealed increased macrophage infiltration compared to controls after BMI stratification. Similarly, mice with kidney disease recapitulated the inflammatory state observed in uremic patients and additionally demonstrated increased peripheral monocytes and inflammatory polarization of adipose tissue macrophages (ATMS). In contrast, adipose tissue in uremic IL-6 knock out mice showed reduced ATMS density compared to uremic wild-type controls. Differences in ATMS density highlight the necessary role of IL-6 in macrophage infiltration in uremia. Uremia promotes changes in adipocytes and macrophages enhancing production of inflammatory cytokines. We demonstrate an interaction between uremic activated macrophages and adipose tissue that augments inflammation in uremia.

GSTM1 Modulates Expression of Endothelial Adhesion Molecules in Uremic Milieu

Deletion polymorphism of glutathione S-transferase M1 (GSTM1), a phase II detoxification and antioxidant enzyme, increases susceptibility to end-stage renal disease (ESRD) as well as the development of cardiovascular diseases (CVD) among ESRD patients and leads to their shorter cardiovascular survival. The mechanisms by which GSTM1 downregulation contributes to oxidative stress and inflammation in endothelial cells in uremic conditions have not been investigated so far. Therefore, the aim of the present study was to elucidate the effects of GSTM1 knockdown on oxidative stress and expression of a panel of inflammatory markers in human umbilical vein endothelial cells (HUVECs) exposed to uremic serum. Additionally, we aimed to discern whether GSTM1-null genotype is associated with serum levels of adhesion molecules in ESRD patients. HUVECs treated with uremic serum exhibited impaired redox balance characterized by enhanced lipid peroxidation and decreased antioxidant enzyme activities, independently of the GSTM1 knockdown. In response to uremic injury, HUVECs exhibited alteration in the expression of a series of inflammatory cytokines including retinol-binding protein 4 (RBP4), regulated on activation, normal T cell expressed and secreted (RANTES), C-reactive protein (CRP), angiogenin, dickkopf-1 (Dkk-1), and platelet factor 4 (PF4). GSTM1 knockdown in HUVECs showed upregulation of monocyte chemoattractant protein-1 (MCP-1), a cytokine involved in the regulation of monocyte migration and adhesion. These cells also have shown upregulated intracellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). In accordance with these findings, the levels of serum ICAM-1 and VCAM-1 (sICAM-1 and sVCAM-1) were increased in ESRD patients lacking GSTM1, in comparison with patients with the GSTM1-active genotype. Based on these results, it may be concluded that incubation of endothelial cells in uremic serum induces redox imbalance accompanied with altered expression of a series of cytokines involved in arteriosclerosis and atherosclerosis. The association of GSTM1 downregulation with the altered expression of adhesion molecules might be at least partly responsible for the increased susceptibility of ESRD patients to CVD.

Modeling Uremic Vasculopathy With Induced Pluripotent Stem Cell-Derived Endothelial Cells as a Drug Screening System

Background: Cardiovascular complications are the leading cause of mortality in patients with chronic kidney disease (CKD). Uremic vasculopathy plays a crucial role in facilitating the progression of cardiovascular complications in advanced CKD. However, the improvement of conventional research methods could provide further insights into CKD.Objectives: In this study, we aimed to develop a novel model of uremic vasculopathy as a potential drug screening system.Methods and Results: The effects of uremic serum and different combinations of uremic toxins on induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) of a normal control and a CKD patient were investigated using several functional assays. We found that a mixture of uremic toxins composed of high urea, creatinine, uric acid, and indoxyl sulfate exerted deleterious effects on normal control iPSC-ECs that were comparable to uremic serum by increasing reactive oxygen species and apoptosis, as well as suppression of tube formation. Additional characterization revealed a potential involvement of dysregulated TGF-β signaling as treatment with either losartan or TGF-β inhibitors led to the attenuation of adverse effects induced by uremic toxins. Importantly, impaired wound healing potential seen in CKD patient-specific iPSC-ECs was rescued by treatment with losartan and TGF-β inhibitors.Conclusion: Our study demonstrated that simplified uremic toxin mixtures can simulate the uremic micromilieu reproducibly and CKD patient-specific iPSC-ECs can potentially recapitulate susceptibility to uremic vasculopathy. This novel model of uremic vasculopathy may provide a new research tool as a drug screening system.