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High salt intake alters renal medullary clock genes via ETB receptors

Diurnal rhythms in various physiological functions are driven by the oscillations of several transcription factors including Bmal1, period (Per), cryptochrome (Cry), and clock, collectively termed the molecular clock. The renal inner medullary collecting duct (IMCD) is an important site for Na+ handling and contains several clock factors. The IMCD also produces ET‐1 in response to high salt intake and contains a high density of endothelin B (ETB) receptors that promote the excretion of Na+. Disruption of renal ETB receptors reduces the ability of kidney to excrete a salt load in rodents, especially when salt is given prior to the inactive period vs. the active period. These data suggest an interaction between renal ETB receptors and the molecular clock to promote the excretion of excess Na+. Given that knockout of the circadian clock gene Bmal1 reduces blood pressure during the active period in mice, we hypothesized that a high salt intake suppresses the clock mechanism in the renal inner medulla via ETB receptor activation. Transgenic control (Tg con) or ETB deficient (ETB def) rats were fed normal (NS, 0.49% NaCl) or high (HS, 4% NaCl) salt for two weeks. Rats were euthanized every 4 hours beginning at zeitgeber time 0 (lights on) for tissue collection and subsequent assessment of circadian clock genes. Renal inner medullary Bmal1 oscillation was phase shifted in Tg con rats in response to HS compared to NS fed rats. On the other hand, Bmal1 expression was similar between NS and HS fed ETB def rats indicating that the phase shift in Tg con rats is ET‐dependent. In addition, renal inner medullary Per1 expression was similar between all groups; however expression of Per2, Cry1, and Cry2 were suppressed during normal acrophase in response to HS in both Tg con and ETB def rats suggesting that this is ET‐independent. Further, in cultured mouse IMCD cells, treatment with ET‐1 significantly reduced Bmal1 mRNA. Finally, knockdown of Bmal1 with siRNA significantly reduced mRNA expression of the epithelial Na+ channel alpha subunit (ENaCα) (1.0±0.06 vs. 0.74±0.07, control vs. Bmal siRNA: p<0.05) and increased transepithelial resistance (583.0±43.3 vs. 703.5±32.6 ohms, control vs. Bmal siRNA: p=0.056) in mouse IMCD cells. These data suggest that activation of renal inner medullary ETB receptors during high salt intake functions to regulate the normal circadian pattern of Bmal1 expression in order to suppress ENaCα and promote the excretion of excess Na+.