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 pressure-lowering 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.
Highlights
ET-1 has many functions including vascular constriction, nervous system activation, and renal sodium secretion
Using a human kidney proximal tubule cell line (HK-2), we show that CRISPRmediated deletion of a regulatory element within the endothlien-1 gene (EDN1)-AS promoter resulted in increased levels of EDN1-AS
Transcription Factor II D (TFIID), Upstream Transcription Factor 1 (USF1), and Glucocorticoid Receptor (GR) are predicted to bind to this region, and these transcription factors are associated with sites of active transcription
Summary
ET-1 has many functions including vascular constriction, nervous system activation, and renal sodium secretion. The kidney is both a source and a target of ET-1, and the kidney has the highest concentrations of ET-1 found in the body (Kohan et al, 2011). The mechanisms of ET1 action in the pathology of chronic kidney disease (CKD) involve increased cell proliferation, inflammation, and elaboration of the extracellular matrix (Speed and Pollock, 2013; Kohan and Barton, 2014; Reichetzeder et al, 2014). Mice overexpressing ET-1 developed chronic renal failure (Theuring et al, 1998). Excess ET-1 levels are seen in all stages of CKD (Dhaun et al, 2009), and there are marked increases in urinary ET-1 secretion in CKD (Grenda et al, 2007).
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