Background
End-stage renal disease (ESRD) is the final stage of chronic kidney disease (CKD), characterized by a glomerular filtration rate (GFR) of less than 15 ml/min/1.73m
2, as well as structural and functional deterioration for at least 3 months [
1]. According to the recent studies, diabetes and hypertension are the primary causes of ESRD in developed countries [
2], whereas the transition from glomerulonephritis to metabolic kidney disorders has become the leading cause in low- and middle-income nations [
3,
4]. The prevalence of ESRD has risen considerably as diabetes and hypertension have become more prevalent [
5,
6]. Currently, kidney replacement therapy (KRT), which includes hemodialysis, peritoneal dialysis, and kidney transplantation, is the most common treatment for ESRD. Global KRT usage is predicted to reach 5.4 million by 2030, with Asia seeing the most growth [
7]. Cardiovascular illnesses, uremia, volume overload, malnutrition, uncontrolled hypertension, cancer, severe infections, and dialysis-related complications are all major causes of death in people with ESRD [
8‐
10]. ESRD has become a global concern since it would significantly diminish life quality and increase financial cost on families and society.
Yasuo Hotta and Alix Bassel discovered extrachromosomal circular DNA (eccDNA) in pig sperm and wheat nuclei in 1964 [
11]. EccDNA is a double-stranded circular DNA that exists in addition to the structurally complete chromosome genomic DNA. Following that, eccDNAs were discovered in human tumor cells [
12] and practically all organisms [
13,
14]. EccDNAs have been classified as micro DNA (100–400 bp) [
15], small polydispersed circular DNAs (spcDNAs, 100 bp–10 kb) [
16], mitochondrial DNAs (mtDNAs, 16 kb) [
17], and double-minutes (DMs, 100 kb–3 Mb) [
17,
18] based on their length and features. Previous research has linked eccDNA to genomic rearrangement, cell apoptosis, episome enlargement, translocation, and amplification [
19‐
22]. Several studies have proclaimed that eccDNA potentially play an important role in disorders involving epigenetic modulation, such as cancer [
23]. EccDNA can cause gene deletion, mutation, duplication, or amplification, resulting in genetic heterogeneity and adaptive evolution between cells. Recent research by lv [
24] examined the physical characteristics of eccDNA, such as length, GC content, and motif signature, etc. and miRNA in urine from CKD patients, and found that the eccDNA count in the CKD group was higher than it was in the healthy group. Although preliminary study on the properties of eccDNA in CKD patients revealed certain differences between the patients and healthy individuals, there is still a dearth of knowledge regarding the role and probable mechanism of eccDNA in CKD, particularly in ESRD patients, which we intended to focus on.
In this study, we used a high-throughput approach, namely Circle-sequencing, to collect and identify eccDNAs from peripheral blood mononuclear cells (PBMCs) of ESRD patients (n = 16) and healthy people (n = 12). We detected 14,431 and 11,324 eccDNAs in the ESRD and NC groups, respectively, with a large range of eccDNA sizes. Furthermore, we identified that the specific eccDNA in ESRD was markedly enriched in cell junction and communication pathways. Out of all the eccDNA-targeting genes specific to ESRD, we discovered potentially 20 hub genes. Additionally, we found that eccDNA-targeting genes, including CCL2, CCR2, MYH9, and IL10, were critical in the development of ESRD, indicating that these eccDNAs had novel biological roles for ESRD patients.
Discussion
Multiple variables have a role in the development of ESRD because it can be exacerbated by the evolution of a range of kidney illnesses. For starters, renal and systemic inflammation are significant causes of ESRD in patients with kidney diseases [
35]. Furthermore, multiple studies have revealed that TFs, DNA, and chromosome damage, as well as metabolites in urine and blood, are all linked to the development of ESRD and can be manifested to be potential diagnostic and prognostic biomarkers [
36‐
38]. However, there are currently no daily clinical examinational biomarkers for ESRD. With advances in technology of sequencing, quantities of latest studies have discovered that eccDNA physically excised from the chromosome is involved in a wide range of biological processes, including cell–cell communication, aging, intercellular genetic heterogeneity, regulating innate immunity, transcribed into noncoding RNAs, and participates in the cancer physiological processes [
39‐
41]. However, the biochemical function of eccDNA in ESRD patients is inadequately defined. As far as we know, this study is the first to illustrate the expression and function of eccDNA in ESRD patients. This study extends the knowledge of the characteristics of eccDNA for ESRD patients. Using the circle-seq technology, we were able to identify eccDNA in PBMCs from ESRD patients and healthy people, as well as reveal the characteristics of eccDNA in PBMCs from patients with ESRD, such as the number of eccDNA, length distribution, genome distribution, motif, and function of genes on eccDNA. Finally, we identified 20 hub genes and 39 ESRD-related genes of ESRD-specific eccDNA-targeting genes.
EccDNA is featured with motifs siding the start and end. Motifs are DNA sequences that provide binding sites for a type of protein called TFs, which govern the activation or repression of gene expression by recognizing motifs found at regulatory regions to regulate downstream chromatin processes [
42]. The major ESRD transcription factor GLIS3 in this study, which belongs to the Krüppel-like zinc finger protein family, is mostly expressed in the kidney, thyroid, and pancreas. GLIS3 has been indicated to act a significant part in preserving the natural structure and function of the kidney as part of transcription regulatory networks, and GLIS3 mutant develops polycystic kidney disease [
43]. However, whether GLIS3 leads to other kidney diseases even ESRD remains room for further research. Furthermore, the transcription factor Egr2 has been demonstrated to play a role in neutrophil degranulation and immunological activation in ESRD patients on nocturnal hemodialysis [
44]. Pitx1, IRF3, and ETS1 are also TFs involved in the pathogenesis of kidney diseases [
45‐
47]. Hence, we conjecture that eccDNA develops ESRD as a result of the TFs mentioned above.
In this work, we discovered 20 top eccDNA hub genes in which NCAM1, NFATC1, PRKCB, LEF1, PRKAG2, and GRM8 were strongly linked to a variety of kidney disorders. For example, gene NFATC1 has been associated with LN [
48], gene PRKCB1 has been linked to the progression from DN to ESRD [
49], transcription factor LEF1 encoded by gene LEF1 engaged in the Wnt signaling pathway is linked to CKD [
50], and gene PRKAG2 is a fresh locus for CKD [
51]. In addition, we searched databases and discovered that some ESRD-specific targeting-genes were known to be related to several kidney diseases involved in this study (Additional file
1: Table S1). Obviously, the majority of ESRD-related genes were linked to DN, CGN, or LN as well, which was consistent with the progression of primary diseases into ESRD. Among them, we noticed that CCL2, CCR2, MYH9, and IL10 were present in all four diseases. According to the reports, monocyte chemoattractant protein 1 (MCP-1) encoded by CCL2 was a biomarker in kidney diseases, suggesting kidney damage and inflammation, and CCL2 itself increased in macrophages and was related to renal fibrosis in a renal atrophy model [
52]. The genotype frequency of polymorphisms in CCR2 and IL10 showed a great difference between ESRD and controls, especially IL10, which demonstrated their susceptibility to ESRD [
53]. MYH9 mutation might disorder renal epithelial transport pathways and further result in kidney diseases [
54]. The above ESRD-related genes and functional analysis in Fig.
5C were displayed that inflammation and renal epithelial cell dysfunction were essential mechanisms in ESRD and implied that eccDNA in these genes played a significant part in the progression of ESRD which was the subject of further research.
According to GO analysis, “regulation of cell shape” is the most enriched biological process among the genes predicted by specifically expressed eccDNA in the ESRD group as compared to the NC group. Planar cell polarity (PCP) refers to the coordinated orientation of cells in the tissue plane. Protein encoded by PCP genes and PCP signaling pathway regulate cell shape and behavior, as well as kidney development and diseases, such as polycystic kidney disease and Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) [
55]. In addition, actomyosin, a prominent cellular target of the PCP signaling pathway, not only regulates cell shape and motility, but it can also be cleaved by activated caspase-3, causing muscle atrophy in patients with CKD [
56]. These findings suggest that eccDNA hub genes may have a role in the pathophysiology of ESRD via regulating cell shape. Furthermore, in comparison to the NC group, "glutamate receptor activity" is the most enriched molecular function of specifically expressed eccDNA in the ESRD group. Ionotropic receptors, such as NMDA receptors, AMPA receptors, and KA receptors, along with metabotropic L-Glu receptors (mGluRs), are the two types of glutamate receptors. The toxicity of overactivated NMDA receptors on renal cells has been demonstrated [
57]. Ca
2+ influx and oxidative stress are caused by sustained NMDA receptors activation, which can contribute to glomerulosclerosis. Ca
2+ influx pathways TRPC6 that amplifies Ca
2+ excess activated by NMDA receptors regulates Rac1 of the Rho protein family to modulate signal transduction that influences a great many aspects of cell behavior, including cytoskeletal dynamics in podocytes [
58]. The abnormality of actin cytoskeleton then breaches the barrier of proteinuria and finally gives rise to CGN, such as focal segmental glomerulosclerosis (FSGS) [
59], which is the most common cause of ESRD and has the most cases in this study, implying that the aforesaid hub genes may be involved in the pathogenic process of ESRD. The primary pathway of ESRD patients with a comparison of healthy individuals in the present study is “phosphatidylinositol signaling system” among the top 20 enrichment KEGG pathways. Phosphatidylinositol signaling pathway is involved in a variety of biological activities, such as cell proliferation, cell differentiation, apoptosis, and membrane trafficking [
60]. Among this signaling system, abnormal activation of the phosphoinositide 3-kinase gamma (PI3Kγ) signaling pathway has been shown to play an important role in the regulation of profibrotic phenotypes. In kidney disease, blocking PI3Kγ signaling pathway in Ang-II-induced kidney damage could alleviate renal injury and fibrosis, and thus improve renal functions, as well as be investigated as a fresh therapeutic method for the treatment of renal fibrosis, renal hypertension, and CKD [
61]. Furthermore, protein-energy wasting characterized by muscle wasting is obviously manifested in ESRD patients, increasing the morbidity and mortality [
62]. A decrease in PI3K activity in skeletal muscle has been shown to aggravate caspase-3 activity and enhance protein degradation, leading to and speeding up wasting through hemodialysis [
63]. This latently indicated that eccDNAs from hub genes could be utilized as biomarkers of diagnosis and progression for ESRD. We look forward to further experimental testimonies to validate these findings.
Despite a bit of advances made by relying on genomic and bioinformatics analysis, it is vital to be acknowledged that there are still limitations to current study. Above important, although the discovery of expression of hub genes on eccDNA in the PBMCs of ESRD patients has been observed by sequencing, there is a paucity of further validation. Additionally, their expression and mechanisms have not yet been authenticated by functional experiments in ESRD. In the next part, fewer cases were included and failed to be gathered by a single primary disease.
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