EDN1-AS, A Novel Long Non-coding RNA Regulating Endothelin-1 in Human Proximal Tubule Cells

Abstract

Endothelin-1 (ET-1) is a peptide hormone that functions as a vasoconstrictor in the vasculature, whereas in the collecting duct of the kidney it exerts blood pressurelowering effects via natriuretic actions. Aberrant ET-1 signaling is associated with several pathological states including hypertension and chronic kidney disease. ET-1 expression is regulated largely through transcriptional control of the gene that encodes ET-1, EDN1. Here we report a long, non-coding RNA (lncRNA) that appears to be antisense to the EDN1 gene, called EDN1-AS. Because EDN1-AS represents a potential novel mechanism to regulate ET-1 expression, we examined the regulation of EDN1-AS expression and action. A putative glucocorticoid receptor response (GR) element upstream of the predicted EDN1-AS transcription start site was identified using the ENCODE database and the UCSC genome browser. Two homozygous deletion clones of the element were generated using CRISPR/Cas9. This deletion resulted in a significant increase in the expression of EDN1-AS, which was associated with increased secretion of ET-1 peptide from HK-2 cells (two-fold increase in KO cells vs. CNTL, n = 7, P < 0.05). Phenotypic characterization of these CRISPR clones revealed a difference in cell growth rates. Using a standard growth assay, we determined that the KO1 clone exhibited a three-fold increase in growth over 8 days compared to control cells (n = 4, P < 0.01) and the KO2 clone exhibited a two-fold increase (n = 4, P < 0.01). These results support a role for EDN1-AS as a novel regulatory mechanism of ET-1 expression and cellular proliferation.

Circadian Gene Expression in Mouse Renal Proximal Tubule

“…Among these, only Rorc showed circadian variation of transcript abundance with peak expression at Zeitgeber 20, https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox/). Note, however, prior studies have provided strong evidence for important roles for components of the classical circadian signaling pathway in regulation of various aspects of kidney function (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)49).…”

“…However, recent studies have reported significant circadian variability in gene expression in the kidney (17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

Role of microRNAs and long non‑coding RNAs in glucocorticoid signaling (Review)

“…EDN1-AS is expressed in multiple human cell types, including the kidneys. In the study by Douma et al (15) in a human kidney proximal tubule cell line (HK-2), the promoter of lncRNA EDN1-AS appeared to have a GRE sequence, which could be recognized and bound by GR, as well as the MR, representing a new mechanism for regulating ET-1 expression. Using CRISPR/Cas9 for the deletion of the GRE element from the EDN1-AS promoter, abolished the binding of GR to EDN1-AS and resulted in increased EDN1-AS expression with a concomitant increase in endothelin 1 expression and cell proliferation.…”

“…Furthermore, miRNAs are directly or indirectly influenced by GCs, while it has been shown that the expression level of miRNAs is dependent on endogenous GC levels (13). In addition, long ncRNAs (lncRNAs), such as growth arrest-specific 5 (GAS5) (14) and EDN1-AS (15), regulate the expression levels of GRs, interfering with the GC signaling pathways. It is therefore paramount to increase our understanding of how nc-RNAs, mainly via the action of miRNAs and lncRNAs, regulate GR and are regulated by GCs, building an intricate regulatory network to fine-tune the body's response to various stimuli.…”

“…EDN1-AS is a lncRNA, which appears to interact with GR. This lncRNA is located antisense of the endothelin 1 gene, which is a peptide hormone that acts on the vascular system as a vasoconstrictor, while in the kidneys it affects blood pressure through diuretic and natriuretic effects (15). Its aberrant quantity has been shown to be associated with pathological conditions, such as hypertension (149) and chronic kidney disease (150).…”