A role for the circadian clock protein Per1 in the regulation of aldosterone levels and renal Na+ retention

Abstract

The circadian clock plays an important role in the regulation of physiological processes, including renal function and blood pressure. We have previously shown that the circadian protein period (Per)1 regulates the expression of multiple Na(+) transport genes in the collecting duct, including the α-subunit of the renal epithelial Na(+) channel. Consistent with this finding, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. We have also recently demonstrated the potential opposing actions of cryptochrome (Cry)2 on Per1 target genes. Recent work by others has demonstrated that Cry1/2 regulates aldosterone production through increased expression of the adrenal gland-specific rate-limiting enzyme 3β-dehydrogenase isomerase (3β-HSD). Therefore, we tested the hypothesis that Per1 plays a role in the regulation of aldosterone levels and renal Na(+) retention. Using RNA silencing and pharmacological blockade of Per1 nuclear entry in the NCI-H295R human adrenal cell line, we showed that Per1 regulates 3β-HSD expression in vitro. These results were confirmed in vivo: mice with reduced levels of Per1 had decreased levels of plasma aldosterone and decreased mRNA expression of 3β-HSD. We postulated that mice with reduced Per1 would have a renal Na(+)-retaining defect. Indeed, metabolic cage experiments demonstrated that Per1 heterozygotes excreted more urinary Na(+) compared with wild-type mice. Taken together, these data support the hypothesis that Per1 regulates aldosterone levels and that Per1 plays an integral role in the regulation of Na(+) retention.

Keywords: aldosterone; circadian clock; kidney; period 1; sodium transport.

Fig. 1.

Decreased period 1 (Per1) results in lower plasma aldosterone levels. Plasma aldosterone levels were determined by ELISA (ENZO). A: plasma aldosterone in wild-type (WT) and Per1 heterozygous (Per1 het) mice at noon and midnight. n = 4–9. B: relative 3β-dehydrogenase isomerase (3β-HSD) mRNA expression from adrenal glands harvested from WT and Per1 het mice at noon and midnight. n = 4–8. WT expression at midnight was set to 100%. C: relative CYPB11 mRNA expression from adrenal glands harvested from WT and Per1 het mice at noon and midnight. WT expression at midnight was set to 100%. n = 4–8. *P < 0.05 compared with WT mice; †P < 0.05 compared with time (noon vs. midnight); ‡P < 0.001, interaction via two-way ANOVA.

Fig. 2.

Per1 knockdown results in decreased 3β-HSD expression in adrenal cells in vitro. NCI-H295R cells were treated with nontarget or Per1 small interfering (si)RNA for 48 h. Quantitative PCR was used to evaluate changes in Per1 (A), 3β-HSD (B), or CYB11 (C) expression in Per1 siRNA versus the nontarget siRNA control. Values are presented as means ± SE; n = 3. *P < 0.05; **P < 0.01.

Fig. 3.

Pharmacological blockade of Per1 nuclear entry results in decreased 3β-HSD expression in vitro. A and B: NCI-H295R cells were treated with the casein kinase (CK)1-δ/ε inhibitor (CKinh) PF-670462 at either 0.1, 1, 10, or 100 μM for 24, 48, or 72 h. Quantitative PCR was used to evaluate changes in 3β-HSD (A) and CYPB11 (B) gene expression in PF-670462-treated versus water-treated cells. Values are presented as means ± SE; n = 3. *P < 0.05; **P < 0.01. C: nuclear extracts were collected from NCI-H295R cells treated with 0.1 μM of the CK-1δ/ε inhibitor PF-670462 or water for 24 h. Western blot analysis was performed using anti-Per1 or anti-β-actin antibodies as a loading control. Data are representative of three independent experiments. D: densitometry analysis was used to quantitate the level of Per1 in C. Values are presented as means ± SE; n = 3. *P < 0.05.

Fig. 4.

Pharmacological blockade of Per1 nuclear entry results in decreased 3β-HSD in vivo. Weight-matched male WT 129/sv mice were given either vehicle (20% hydroxypropyl-β-cyclodextrin) or 30 mg/kg PF-670462 subcutaneously every 12 h for 2.5 days starting at noon and euthanized at midnight 12 h after the last injection, as previously published and described (27). Adrenal glands were harvested, and 3β-HSD (A) and CYPB11 (B) mRNA expression was measured by quantitative PCR. Values are presented as means ± SE; n = 4. *P < 0.05 compared with WT mice.

Fig. 5.

Reduction of Per1 expression in vitro and in vivo results in increased cryptochrome (Cry)2 mRNA in adrenal cells and tissue. NCI-H295R cells were treated with nontarget or Per1 siRNA for 48 h. Quantitative PCR was used to evaluate changes in Cry2 expression in Per1 siRNA versus the nontarget siRNA control. Values are presented as means ± SE; n = 3. *P < 0.05. B: NCI-H295R cells were treated with 0.1 μM of the CK1-δ/ε inhibitor PF-670462 for 24 h. Quantitative PCR was used to evaluate changes in Cry2 gene expression in PF-670462-treated versus water-treated cells. Values are presented as means ± SE; n = 3. *P < 0.05. C: relative Cry2 mRNA expression from adrenal glands harvested from WT and Per1 het mice at midnight. n = 4–8. **P < 0.01 compared with WT mice. D: weight-matched male WT 129/sv mice were given vehicle (20% hydroxypropyl-β-cyclodextrin) or 30 mg/kg PF-670462 subcutaneously every 12 h for 2.5 days starting at noon and euthanized at midnight 12 h after the last injection as previously described (27). Adrenal glands were harvested, and Cry2 expression was measured by quantitative PCR. Values are presented as means ± SE; n = 4. *P < 0.05 compared with WT mice.

Fig. 6.

Decreased Per1 results in impaired renal Na⁺ conservation. Weight-matched male WT 129/sv and Per1 het mice were placed in metabolic cages and allowed to acclimate for 3 days. Per1 het mice were fed the same amount of food as the WT mice ate the previous day. Mice were fed a normal gel diet (0.2% Na⁺) with free access to water. After 3 days, mice were switched to a low-Na⁺ diet (0.02%) for 7 days. Urine samples were collected daily. Flame photometry was performed to determine urinary Na⁺ concentrations. A: urinary Na⁺ retention over time with the average urinary Na⁺ retention during the normal diet and low-Na⁺ diet. n = 5. *P < 0.05 compared with WT mice on given day. B: data shown by genotype and diet, averaged over the course of the normal diet (days 1–3) versus the low-Na⁺ diet (days 4–10). n = 5. *P < 0.05 compared with WT mice; †P < 0.05 compared with time (noon vs. midnight); ‡P < 0.01, significant interaction by two-way ANOVA. C: body weight relative to starting weight (day 3 of the acclimation period). n = 5. *P < 0.05 compared with control.

Fig. 7.

Per1 het mice excrete less Na⁺ in their feces on normal and low-Na⁺ diets. Weight-matched male WT 129/sv and Per1 het mice were placed in metabolic cages and allowed to acclimate for 3 days. Per1 het mice were fed the same amount of food as the WT mice ate the previous day. Mice were fed a normal gel diet (0.2% Na⁺) with free access to water. After 3 days, mice were switched to a low-Na⁺ diet (0.02%) for 7 days. Feces were collected daily. Flame photometry was performed to determine fecal Na⁺ concentrations on day 3 (normal diet) and day 10 (low-Na⁺ diet). Fecal Na⁺ excretion from day 3 (normal diet) and day 10 (low-Na⁺ diet) is shown. n = 6. *P < 0.05 compared with WT mice; †P < 0.05 compared with time (noon vs. midnight).

Fig. 8.

Decreased Per1 results in decreased urinary aldosterone levels. Weight-matched male WT 129/sv and Per1 het mice were placed in metabolic cages and allowed to acclimate for 3 days. Per1 het mice were fed the same amount of food as the WT mice ate the previous day. Mice were fed a normal gel diet (0.2% Na⁺) with free access to water. After 3 days, mice were switched to a low-Na⁺ diet (0.02%) for 7 days. Urinary aldosterone levels were determined by ELISA (ENZO) in WT and Per1 het mice on normal (day 3) and low-Na⁺ (day 10) diets. n =5. *P < 0.05 compared with WT mice; †P < 0.05 compared with diet (normal vs. low Na⁺).