In this study, we used microarray approach to identify HCAEC gene expression signatures of ESRD with and without MI. Previous literature has revealed that there is an intrinsic interplay between ESRD and CVD, while the detailed mechanism remains unclear. We report the gene expression profile obtained from uremic serum stimulation of endothelial cells in an in vitro model. We also identify common molecular pathways linked to important physiological processes. According to the GO classification, we found that genes differentially express a variety of transcription factors that are involved in the immune response. Several studies have discussed the role of inflammation as a first step to promote endothelial dysfunction and progression of atherosclerotic processes [27]
There are studies that suggest that atherosclerosis could be caused by an immune reaction against autoantigens such as oxidized low-density lipoproteins (LDL) and heat shock proteins (HSP) [27]. Interestingly our microarray profile highlights some genes that could sustain common molecular alterations in ESRD and ECV. Some of these genes, but not all, were independently validated by RT-PCR analysis on the samples.
The cardiovascular system is the main target of uremic toxins and chronic inflammation in ESRD patients. Genetic studies with focus in endothelial dysfunction associated to cardiovascular develop are scarce. The main purpose of this study was to explore the effect of uremic serum on the gene expression pattern of HCAEC associated to adverse cardiovascular outcomes in CKD patients through a microarray analysis.
Although it is widely recognized that patients on dialysis have substantially higher cardiovascular and non-cardiovascular mortality rates compared with the general population, little is known about the genetic predisposition to mortality of these vulnerable patients. In the present study, we investigated serum of patients with ESRD have a very high mortality risk as compared with the general population. Cardiovascular disease is a major cause of death in these patients, accounting for 40–50% of total mortality [28, 29].
Currently, CKD is associated with an increased risk of CVD. In ESRD patients, CVD is responsible for almost 50% of deaths [30]. Several studies have focused on clearing out the mechanisms involved in the increase of the risk. Uremic toxins have been classified into three major groups as proposed by the European Uremic Toxin Work Group (EUTox) as well as their behavior during dialysis in: a. Water-soluble molecules of low molecular weight, such as urea; b. Middle molecules; and c. Protein-bound uremic toxins whose removal through conventional dialysis and hemodialysis treatments is problematic due to their high protein (mostly albumin)-binding affinity [31].
Endothelium, a disseminated organ, is a major component of most organs. Due to its disseminated nature and involvement in the normal physiology in the body it has a myriad of functions. Unsurprising, this functional heterogeneity requires not only a high variation in its phenotype expression which depends on the vascular bed but also needs the ability to react according to the environs in health and disease. We may appreciate the importance of EC by the fact that it is involved in almost all disease states either as a primary determinant or as an innocent bystander [32].
A permanent EC aggression as a result of chronic exposure to uremic toxins induces cellular phenotype abnormalities which may result in high serum levels of inflammatory biomarkers such as IL-8 and MCP-1 (CCL2), cytokines, and the adhesion moleculesVCAM-1 and ICAM-1 [33]. Serum levels of these entire biomarkers rise in patients with CKD a fact that suggests a link between vascular activation, inflammation, and uremic toxicity [34]. Uremic toxins have been associated with EC dysfunction in CKD patients. As a consequence, the uremic toxins may induce active free radicals [34]. Uremic toxins and chronic inflammation undoubtedly contribute to EC dysfunction associated to the CV but the complex mechanism associated to CKD alterations needs to be more elucidated. EC have multiple functions such as regulation of hemodynamics, permeability, nutrients exchange, leukocyte interaction, and blood coagulation, amongst others. Chronic EC dysfunction is also considered as the main event in atherosclerosis which progresses toward a pro-inflammatory cell pattern, senescence, and apoptosis [10].
Moreover, accumulation of uremic toxins induces oxidative stress (OS) related to reactive oxygen species and reactive nitrogen species production (RONS) [35], which, in the vessel wall are mainly produced from NADPH oxidase, xanthine oxidase, the mitochondrial respiratory chain, and uncoupled endothelial nitric oxide synthase (eNOS) in which oxygen is reduced from nitric oxide (NO) synthesis [35]. In a patient with ESRD uremic toxins promote vascular leakage by increasing EC permeability, impaired blood flow, and leukocyte adhesion [36]. Moreover, they may have pro-fibrotic and pro-hypertrophic effects on cardiac cells as well as a proinflammatory effects in monocytes by increasing gene expression of key inflammatory cytokines involved in the progression of heart failure [37]. The cardiac profibrotic effect of these toxins are also observed in patients with renal failure and in animal models of MI with concomitant renal impairment which is likely mediated through the oxidative stress/NF-ĸB/TGF-β pathway [38]. Furthermore, the proteomic approaching in ESRD patients reveals changes in the expression of inflammation and oxidative stress related molecules; some of these changes correlated with NFkB activation [39]. Additionally, in animal models, elevated IL-18 levels are associated with pressure overload and inflammatory states and may play a role in cardiac hypertrophy and remodeling [40].
This methodology also revealed possible mechanisms involved in ESRD in patients with EC dysfunction and showed six genes involved in the regulation of cell-cycle progression (CDK-1, topoisomerase II, PDZ-binding kinase, CDCA1, protein SDP35, E2F transcription factor 8), and two genes of the cholesterol efflux system (ABCA1 and ABCG1), which were down-regulated in HCAECs exposed to uremic plasma [19].
We used a microarray technology to investigate the gene expression profiles in HCAECs, induced by serum from USI and UCI patients and we explored which pathways were potentially involved in this process. This microarray approach allowed us to reduce biases due to the relatively small number of patients selected and to minimize confounding factors. Microarray analysis was performed in an UCI group to assess differences in the gene expression pattern vs. USI patients. Unsupervised analysis clearly separated the groups demonstrating their differences. Microarray analysis revealed a 100-gene profile differentially expressed which discriminated CKD patients. By enrichment analysis we reduced the set to 50 genes: 30 genes were over-expressed and 12 were under-expressed in the UCI group. Although we identified candidate molecular markers, it is necessary to test these candidates in independent cohorts before any conclusion concerning their diagnostic impact [40].
The main finding of this work was the identification of key genes involved in the MAPK signaling pathway. The MAPKs signaling pathway is involved in a repertoire of biological events including proliferation, differentiation, metabolism, motility, survival, and apoptosis. And it pathway encompasses a large number of serine/threonine kinases and it is divided into four MAPK subfamilies including extracellular signal-regulated kinases (ERK1/2), c-Jun NH2-terminal kinases (JNK1, -2 and − 3), p38 kinase (α, β, γ, δ,), and big MAPK (BMK or ERK5) [41]. Studies have shown that MAPK subfamilies are involved in the pathogenesis of numerous renal diseases, including CKD and ESRD [42], and produce important signaling molecules involved in inflammatory process in the kidney [48]. Also, previous studies have focused on TGF‑β and epithelial or EC for mesenchymal transition in myofibroblast transformation, which leads to fibrosis [43].
On the other hand, while we looked for a gene-set associated to MAPK signaling pathway, we found a group of four specific DEGs genes members of this pathway: PLA2G4A, IL1A, RASGRP3 and DDIT3, which are molecules related to inflammation, apoptosis, signal transduction and atherosclerosis. PLA2G4A was one of the two most significantly overexpressed genes. Phospholipases A2 (PLA2s), a family of enzymes that hydrolyze the fatty acid at the sn-2 position of phospholipids, play pivotal roles in cell signaling and inflammation [44]. Recently, it has been reported that these enzymes also function as key regulators of lipid droplet (LD) homeostasis [45]. Although various cellular PLA2s may contribute to generating free fatty acids from membrane phospholipids initially needed for LD synthesis, strong evidence supports that the PLA2 form, such as PLA2G4A, is also involved in ER phospholipids remodeling and LD expansion processes [44, 45]. IL1A was also significantly overexpressed in this analysis. This gene codifies for Interleukin-1 (IL-1), a proinflammatory cytokine, plays a crucial role in ischemic stroke (IS) [46]. Because intracranial atherosclerosis is a risk factor for IS [47], this finding strongly suggests that IL-1 is implicated in the pathophysiology of IS. In our study, RAS guanyl nucleotide-releasing protein 3 (RASGRP3), was one of the main over-expressed genes associated with MAPK pathway. Members of the RAS subfamily of GTPases function as signal transductions, like GTP/GDP-regulated switches that cycle between inactive GDP- and active GTP-bound states, serve as RAS activators by promoting acquisition of GTP to maintain the active GTP-bound state, and are the key link between cell surface receptors and RAS activation [48]. DNA damage-inducible transcript 3 (DDIT3), was also significantly overexpressed in this analysis. This gene encodes to a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. The protein functions as a dominant-negative inhibitor by forming heterodimers with other C/EBP members, such as C/EBP and LAP (liver activator protein) and preventing their DNA binding activity. During endoplasmic reticulum stress (such as in pancreatic beta cells or in atherosclerosis associated macrophages), CHOP can induce activation of Ero1, causing calcium release from the endoplasmic reticulum into the cytoplasm, resulting in apoptosis activation [49]. CHOP also induces apoptosis during endoplasmic reticulum stress by growth arrest and DNA damage-inducible protein GADD34 activation [62]. A recent study, which showed a significantly increased DDIT3 protein (ddit3) expression, induced by the exposure of longer MWCNTs [50]. ddit3 is a transcription factor that could regulate a number of inflammatory cytokines, such as IL-6 [50]. Interestingly, the biological network generated by the String software platform showed an important functional role in all processes described previously. Finally, our results revealed a certain genetic profile with a small set of genes which, in the future, could provide additional information about the biological basis of CVD in CKD.
Limitations of the Study
The main limitation of the present pilot study was the number of samples and microarrays analyzed. However, all measurements were performed in duplicate and confirmed by Quantitative reverse transcription PCR (RT-qPCR). The in vitro studies were performed in triplicate in order to minimize variations and confirmed by different experimental approaches.
Our study has limitations because we cannot disregard that several differentially expressed genes were actually derived from contaminating like others serum toxins, particularly environmental pollutants. Therefore, purification of the serum toxins by different methodologies should be implemented to validate all potential biomarkers. Studies in clinical samples to additional validation on independent samples seem necessary.