Loss of the Circadian Protein PER1 Affects Gene Expression in the Adrenal Glands of C57BL/6 Mice Following High Salt Diet Plus Mineralocorticoid Treatment

Abstract

IntroductionThe circadian clock is found in every cell of the body and plays a vital role in various physiological functions. At the molecular level, it is governed by four core transcription factors, PERIOD (PER1), BMAL, CRYPTOCHROME (CRY1/2), and CLOCK. Our lab is interested in the kidney clock and the role it plays in the regulation of blood pressure (BP). Throughout a 24‐hour cycle, BP exhibits a circadian rhythm, peaking during the middle of the active period and then decreasing, or “dipping”, during the middle of the inactive period. Lack of this characteristic dip in BP can lead to non‐dipping hypertension (HTN) and has been associated with an increased risk in adverse cardiovascular events. We have previously shown that male mice lacking the circadian gene Per1 (Per1 KO) develop non‐dipping HTN when given a high salt diet plus mineralocorticoid (HS/DOCP) treatment. This treatment mimics a low‐renin high‐aldosterone model of salt‐sensitive HTN. Additionally, we have observed that at baseline, these male Per1 KO mice exhibit increased levels of urinary aldosterone, a mineralocorticoid hormone derived from the renin‐angiotensin‐aldosterone system (RAAS) in the adrenal glands which promotes sodium reabsorption by the kidneys, leading to increased BP.GoalGiven the close interplay of the adrenal glands and the kidneys, the goal of this study was to determine if administration of a HS/DOCP diet would cause differences in gene expression of circadian (Bmal1, Clock, Cry1/2, and Reverba) and aldosterone synthesis (HSD3B1 and CYP11B) genes in PER1 KO mice compared to wild‐type (WT).MethodsAdrenal glands were obtained at noon (midpoint of mouse’s rest period) from male WT and Per1 KO mice on a control diet or 4 days after HS/DOCP. Total RNA was isolated using Trizol reagent and converted to cDNA. QPCR was performed using TaqMan reagents and beta‐actin as a control. Data was compared by two‐way ANOVA to test for genotype and treatment effects (n=4–6 mice per group).ResultsIt was observed that Bmal1 and CYP11B expression significantly decreases when given HS/DOCP diet compared to control (pBmal1 < 0.05, pCYP11B < 0.0001) whereas expression of Clock and HSD3B1 significantly increases (pClock < 0.05, pHSD3B1 < 0.01). Statistically significant differences were not observed for Cry2. Cry1 expression was significantly suppressed in WT by ~50% after HS/DOCP diet, but there was no effect of HS/DOCP in Per1 KO mice (pgenotype = 0.0035, ptx = 0.0028, pinteraction = 0.0018). The most dramatic difference observed was a five‐fold increase in expression of the circadian gene product Reverba in WT after HS/DOCP treatment, but not in Per1 KO mice (pgenotype < 0.0001, ptx < 0.0001, pinteraction = 0.0003).ConclusionsThese findings support a key role for PER1 in the regulation of Cry1 and Reverba in the adrenal gland. In the future we plan to compare our current findings to female Per1 KO mice in order to determine if there is a sex difference in the regulation of the clock and the aldosterone synthesis pathway.Support or Funding InformationNIH R01DK109570 to MG. AHA postdoc fellowship 18POST34030210 to LD.