Interplay of the Circadian Clock and Endothelin System.

The peptide hormone endothelin-1 and its receptors are linked to several disease states. Pharmacological inhibition of this pathway has proven beneficial in pulmonary hypertension, yet its potential in other disease states remains to be realized. This review considers an often understudied aspect of endothelin biology, circadian rhythm regulation and how understanding the intersection between endothelin signaling and the circadian clock may be leveraged to realize the potential of endothelin-based therapeutics.

Kidney-specific KO of the circadian clock protein PER1 alters renal Na+ handling, aldosterone levels, and kidney/adrenal gene expression.

PERIOD 1 (PER1) is a circadian clock transcription factor that is regulated by aldosterone, a hormone that increases blood volume and Na+ retention to increase blood pressure. Male global Per1 knockout (KO) mice develop reduced night/day differences in Na+ excretion in response to a high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP), a model of salt-sensitive hypertension. In addition, global Per1 KO mice exhibit higher aldosterone levels on a normal-salt diet. To determine the role of Per1 in the kidney, male kidney-specific Per1 KO (KS-Per1 KO) mice were generated using Ksp-cadherin Cre recombinase to remove exons 2-8 of Per1 in the distal nephron and collecting duct. Male KS-Per1 KO mice have increased Na+ retention but have normal diurnal differences in Na+ excretion in response to HS + DOCP. The increased Na+ retention is associated with altered expression of glucocorticoid and mineralocorticoid receptors, increased serum aldosterone, and increased medullary endothelin-1 compared with control mice. Adrenal gland gene expression analysis revealed that circadian clock and aldosterone synthesis genes have altered expression in KS-Per1 KO mice compared with control mice. These results emphasize the importance of the circadian clock not only in maintaining rhythms of physiological functions but also for adaptability in response to environmental cues, such as HS + DOCP, to maintain overall homeostasis. Given the prevalence of salt-sensitive hypertension in the general population, these findings have important implications for our understanding of how circadian clock proteins regulate homeostasis.NEW & NOTEWORTHY For the first time, we show that knockout of the circadian clock transcription factor PERIOD 1 using kidney-specific cadherin Cre results in increased renal Na+ reabsorption, increased aldosterone levels, and changes in gene expression in both the kidney and adrenal gland. Diurnal changes in renal Na+ excretion were not observed, demonstrating that the clock protein PER1 in the kidney is important for maintaining homeostasis and that this effect may be independent of time of day.

Differences in renal BMAL1 contribution to Na+ homeostasis and blood pressure control in male and female mice.

The renal circadian clock has a major influence on the function of the kidney. Aryl hydrocarbon receptor nuclear translocator-like protein 1 [ARNTL; also known as brain and muscle ARNT-like 1 (BMAL1)] is a core clock protein and transcription factor that regulates the expression of nearly half of all genes. Using male and female kidney-specific cadherin BMAL1 knockout (KS-BMAL1 KO) mice, we examined the role of renal distal segment BMAL1 in blood pressure control and solute handling. We confirmed that this mouse model does not express BMAL1 in thick ascending limb, distal convoluted tubule, and collecting duct cells, which are the final locations for solute and fluid regulation. Male KS-BMAL1 KO mice displayed a substantially lower basal systolic blood pressure compared with littermate control mice, yet their circadian rhythm in pressure remained unchanged [male control mice: 127 ± 0.7 mmHg (n = 4) vs. male KS-BMAL KO mice: 119 ± 2.3 mmHg (n = 5), P < 0.05]. Female mice, however, did not display a genotype difference in basal systolic blood pressure [female control mice: 120 ± 1.6 mmHg (n = 5) vs. female KS-BMAL1 KO mice: 119 ± 1.5 mmHg (n = 7), P = 0.4]. In addition, male KS-BMAL1 KO mice had less Na+ retention compared with control mice in response to a K+-restricted diet (15% less following 5 days of treatment). However, there was no genotype difference in Na+ handling after a K+-restricted diet in female mice. Furthermore, there was evidence indicating a sex-specific response to K+ restriction where female mice reabsorbed less Na+ in response to this dietary challenge compared with male mice. We propose that BMAL1 in the distal nephron and collecting duct contributes to blood pressure regulation and Na+ handling in a sex-specific manner.

Knockout of the circadian clock protein PER1 results in sex-dependent alterations of ET-1 production in mice in response to a high-salt diet plus mineralocorticoid treatment.

Previously, we showed that global knockout (KO) of the circadian clock transcription factor PER1 in male, but not female, mice fed a high-salt diet plus mineralocorticoid treatment (HS/DOCP) resulted in nondipping hypertension and decreased night/day ratio of sodium (Na) excretion. Additionally, we have shown that the endothelin-1 (ET-1) gene is targeted by both PER1 and aldosterone. We hypothesized that ET-1 would exhibit a sex-specific response to HS/DOCP treatment in PER1 KO. Here we show that male, but not female, global PER1 KO mice exhibit a decreased night/day ratio of urinary ET-1. Gene expression analysis revealed significant genotype differences in ET-1 and endothelin A receptor (ETA) expression in male, but not female, mice in response to HS/DOCP. Additionally, both wild-type and global PER1 KO male mice significantly increase endothelin B receptor (ETB) expression in response to HS/DOCP, but female mice do not. Finally, siRNA-mediated knockdown of PER1 in mouse cortical collecting duct cells (mpkCCDc14) resulted in increased ET-1 mRNA expression and peptide secretion in response to aldosterone treatment. These data suggest that PER1 is a negative regulator of ET-1 expression in response to HS/DOCP, revealing a novel mechanism for the regulation of renal Na handling in response to HS/DOCP treatment.

Apparent Absence of BMAL1-Dependent Skeletal Muscle-Kidney Cross Talk in Mice.

BMAL1 is a core mammalian circadian clock transcription factor responsible for the regulation of the expression of thousands of genes. Previously, male skeletal-muscle-specific BMAL1-inducible-knockout (iMS-BMAL1 KO) mice have been described as a model that exhibits an aging-like phenotype with an altered gait, reduced mobility, muscle weakness, and impaired glucose uptake. Given this aging phenotype and that chronic kidney disease is a disease of aging, the goal of this study was to determine if iMS-BMAL1 KO mice exhibit a renal phenotype. Male iMS-BMAL1 KO and control mice were challenged with a low potassium diet for five days. Both genotypes responded appropriately by conserving urinary potassium. The iMS-BMAL1 KO mice excreted less potassium during the rest phase during the normal diet but there was no genotype difference during the active phase. Next, iMS-BMAL1 KO and control mice were used to compare markers of kidney injury and assess renal function before and after a phase advance protocol. Following phase advance, no differences were detected in renal mitochondrial function in iMS-BMAL1 KO mice compared to control mice. Additionally, the glomerular filtration rate and renal morphology were similar between groups in response to phase advance. Disruption of the clock in skeletal muscle tissue activates inflammatory pathways within the kidney of male mice, and there is evidence of this affecting other organs, such as the lungs. However, there were no signs of renal injury or altered function following clock disruption of skeletal muscle under the conditions tested.

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

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.