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

Abstract

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.

INTRODUCTION

It is well documented that various physiological functions, such as the sleep/wake cycle, blood pressure (BP), and hormone secretion, exhibit circadian rhythms (1–3). Intrinsic circadian clocks evolved to regulate physiological functions so organisms could anticipate and adapt to the different phases of a 24-h cycle (4, 5). The central circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus and the other organ circadian clocks, referred to as the peripheral clocks, synchronize through neuronal and hormonal signals in response to environmental cues, such as light, food intake, and exercise (6). These signals regulate the activation of the molecular circadian clock, which contains the transcription factors BMAL1, CLOCK, CRYPTOCHROME (CRY), and PERIOD (PER). The molecular clock is present in nearly every cell and acts in feedback loops to dictate their own expression in addition to the expression of thousands of other clock target genes in a tissue-specific manner (7, 8). BMAL1 and CLOCK form a heterodimer and bind to enhancer box (E-box) promoter response elements to enhance transcription of target genes, including PER and CRY, which act as the negative arm of the circadian feedback loop by inhibiting the actions of BMAL1/CLOCK (5). Our laboratory is specifically interested in the role of PER1, a homolog of PER, in the regulation of kidney gene expression and function.

Kidney functions, such as tubular reabsorption, exhibit circadian oscillations (9, 10). Work by our laboratory and others have shown that the kidney circadian clock plays a significant role in the regulation of Na⁺ excretion rhythms. We were the first to show that Per1 transcription is stimulated by aldosterone, a hormone that is known to regulate Na⁺ balance (11). Male global Per1 knockout (KO) C57BL/6J mice exhibited a reduced night/day difference in Na⁺ excretion after acute treatment of a high-salt (HS) diet plus desoxycorticosterone pivalate (DOCP), a long-lasting aldosterone analog (12). HS diet + DOCP treatment (HS + DOCP) mimics the low-renin, high-aldosterone state often observed in human cases of salt-sensitive hypertension, especially in African Americans (13–18). In addition, male global Per1 KO mice also have increased aldosterone at baseline (19). A preliminary study (12) with male kidney-specific Per1 KO (KS-Per1 KO) mice using Ksp-cadherin Cre recombinase revealed that expression of the NaCl cotransporter (NCC) and the α-subunit of the epithelial Na⁺ channel (ENaC) was significantly increased at baseline compared with Cre negative control mice (12).

The objective of this study was to determine the role of PER1 in the kidney on renal Na⁺ handling. Unlike global Per1 KO mice, KS-Per1 KO mice maintained diurnal Na⁺ excretion patterns even after HS + DO CP treatment. Interestingly, KS-Per1 KO mice retained significantly more Na⁺ than control mice, demonstrating the importance of PER1 in the kidneys for maintaining Na⁺ homeostasis. KS-Per1 KO mice also have increased serum aldosterone and medullary endothelin-1 (ET-1) compared with control mice. KS-Per1 KO mice do not downregulate renal glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) expression in response to HS + DOCP, and adrenal gland gene expression analysis revealed increased expression of aldosterone synthase. Our results demonstrate that PER1 is not only important in the regulation of renal Na⁺ handling but also for proper communication between the kidney and adrenals for maintaining overall homeostasis.

METHODS

Mice

All experiments involving animals were approved by the University of Florida and the North Florida/South Georgia Veterans Administration Institutional Animal Care and Use Committees in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Kidney distal nephron-specific C57BL/6J Per1 KO (KS-Per1 KO) mice were created using Ksp-cadherin Cre recombinase (12). Homozygous floxed Per1 mice were generated by the KOMP Repository and crossed with Flp recombinase transgenic mice to remove the neomycin cassette (University of California-Davis Mouse Biology Project) and then transferred to the University of Florida, where they were successfully crossed with Ksp-cadherin Cre mice (Jackson Laboratory) at the University of Florida. Deletion of exons 2–8 in the KS-Per1 KO mice inner medulla was confirmed through recombination PCR.

Recombination PCR

Genomic DNA from the dissected inner medulla (containing the thin limbs, vasa recta, and inner medullary collecting duct) was prepared and used as the template for PCR analysis to show successful recombination and subsequent deletion of exons 2–8. In addition, genomic DNA was isolated from the brain, heart, aorta, adrenal, liver, and lung of the same mice to show that the recombination and deletion of exons 2–8 were specific to kidney tissue. PCR using the forward primer (5′- GTTATGGTGGGTGTTCTTCTCC-3′) and loxP reverse primer (5′- CTAGAACAGTAGCTGAGGGTCAAAG-3′) resulted in a 705-bp product encompassing a loxP site in both intact and recombined Per1 genes. PCR using the forward primer and the recomb reverse primer (5′- GCGAATAGGGGAATGGTCAAAG-3′) resulted in a 1,318-bp product only in KS-Per1 KO mouse kidney tissue due to recombination and removal of the sequence in between the two loxP sites by Cre recombinase. In tissue without recombination between the two loxP sites, the theoretical PCR product using the forward primer and recomb reverse primer is 3,751 bp, but because the PCR extension time is 1 min, this product is not seen in samples where recombination did not occur.

Per1 RNAscope

Mice were anesthetized with inhalant isoflurane. The right kidney was removed, transversely bisected, fixed in a periodate-lysine-paraformaldehyde solution for 48 h, and then embedded in paraffin. Two 4-µm slices were cut and mounted on a glass slide. The method of in situ hybridization was performed using the RNAscope 2.5 HD detection kit according to the manufacturer’s protocols for kidney tissue (Cat. No. 322360, Advanced Cell Diagnostics). In brief, the slide was baked, deparaffinized, rehydrated, and incubated with target retrieval solution for 30 min in a steam bowl. Several steps were carried out to hybridize and amplify target mRNA using a Per1-specific probe (Mm-Per1 no. 438751). Hematoxylin Solution Gill No. 1 (Sigma-Aldrich) was used as a counterstain. The distinct punctate of red chromogen precipitate was examined using bright-field microscopy. Three nonoverlapping representative images of each kidney region (cortex, outer medulla, and inner medulla) were taken at ×40 magnification (Nikon E600 equipped with a Nikon DXM1200F digital camera). A researcher blinded from the hypothesis and animal identification numbers quantified the Per1-positive dots of each image using ImageJ. The red stain was identified and labeled in the image. The count of positively labeled punctate of sizes 11–30 pixels² was recorded. The number of punctate per screenshot under ×40 magnification was converted to punctate per mm² using a total area of 46,875 µm². This process was repeated for each mouse.

Metabolic Cage HS + DOCP Experiments

Male C57BL/6J KS-PER1 KO and control mice of an age of 12 wk were treated as previously described (20). After acclimation to metabolic cages for 3 days, mice were given a control diet (Envigo Teklad custom diet TD.99131) for 3 days with baseline urine collections made on the third day. Twelve-hour urine collections (6 AM and 6 PM) were made throughout the experiments. Mice were then given a HS (4% NaCl, Envigo Teklad custom diet TD.170601) gel diet for a total of 6 days with ad libitum access. On the third day of the HS diet, right before the beginning of the mouse active period (6 PM), mice received an intramuscular injection with 70 µg/g body wt DOCP (Novartis) as previously described (12, 20). Urine was collected for 3 days with HS + DOCP treatment. On the fourth day of HS + DOCP treatment, tissues were harvested at noon.

Flame Photometry

Flame photometry (model 2655-00, Cole-Parmer, Chicago, IL) was used to determine urine and serum electrolyte concentrations. Serum was collected into a heparin-coated syringe using cardiac puncture.

Terminal BP Measurements

Under continuous isoflurane anesthesia with heat support, mice underwent a terminal surgery during which a PAC10 radiotelemeter was used to record BP. The telemeter cannula was placed in the left carotid artery with the tip of the cannula in the free-flowing blood of the aortic arch. BP was measured every 10 s for a total of 10 min, and the average of the measurements was used for analysis. Measurements were performed around noon on for normal-salt (NS) diet, HS diet, and day 3 HS + DOCP groups.

Aldosterone ELISA

Serum aldosterone peptide was measured by ELISA from Enzo (ADI-900-173) according to the manufacturer’s instructions. The cross-reactivity for this kit was 100% for aldosterone, 0.3% for 11-deoxycorticosterone, 0.19% for corticosterone, 0.20% for progesterone, and <0.001% for cortisol, dihydrotestosterone, estradiol, and testosterone. The detectable range was from 3.9 to 250 pg/mL.

ET-1 ELISA

Urinary ET-1 peptide was measured by ELISA from R&D Systems (Endothelin-1 QuantiGlo ELISA kit). This ELISA kit detects both full-length ET-1 and processed ET-1. The cross-reactivity for this kit was 51% for ET-2, 0.01% for full-length ET-2, and 9% for ET-3. The detectable range was from 0.064 to 250 pg/mL. ELISA was performed according to the manufacturer’s instructions with undiluted urine samples after centrifugation to remove debris or with 80 µg of kidney medulla total protein isolated using T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific). Protein isolate concentrations were determined by BCA (Pierce).

RNA Isolation and Real-Time Quantitative RT-PCR

The kidneys and adrenals were removed from the mice at noon, the midpoint of the mouse inactive period, following either a NS diet or 4 days of HS + DOCP. Kidneys were dissected into the cortex and whole medulla. Total RNA was isolated using TRIzol (Invitrogen). RNA was treated with DNaseI (Ambion). The resulting RNA samples were used along with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) to create cDNA. Applied Biosystems TaqMan probes were used for gene expression analysis as previously described (21).

Statistics

Power analysis was based on pilot data obtained for urinary Na⁺ excretion data (12) using SigmaPlot software, with n = 6 calculated to have >85% power, a main effect α level = 0.05, with two experimental groups, SD = 0.1, and difference in means = 0.2. Graphpad Prism was used to perform t tests, two-way ANOVA with Šídák multiple comparisons post hoc analysis, and two-way ANOVA with repeated measures with Šídák multiple comparisons post hoc analysis to determine the effect of treatment (NS, HS, and HS + DOCP) and genotype. All values are presented as means ± SE.

RESULTS

Ksp-Cadherin Cre Recombinase KO of Per1 Is Localized to the Kidney

To confirm Ksp-cadherin Cre KO of Per1 is localized to the kidney, recombination PCR was performed on genomic DNA isolated from the kidney (inner medulla), adrenals, brain, liver, lung, heart, and aorta (Fig. 1B and Supplemental Fig. S1; all Supplemental Material is available at https://doi.org/10.6084/m9.figshare.18416846.v1). Recombination bands were only present in PCRs using genomic DNA from the kidney of Ksp-cadherin Cre positive mice (KS-Per1 KO) but not in PCRs using control genomic DNA or KS-Per1 KO genomic DNA from the brain, liver, lung, heart, aorta, or adrenals. To confirm that Ksp-cadherin Cre KO of Per1 resulted in a reduction of Per1 mRNA, RNAScope was performed on renal inner medulla, outer medulla, and cortex sections isolated from KS-Per1 KO and control mice (Fig. 1C and Supplemental Fig. S2). The presence of Per1 mRNA was significantly reduced in the inner and outer medulla of KS-Per1 KO mice compared with control mice (∼80% decrease, P < 0.01). There was no significant difference in Per1 mRNA in the renal cortex. These results are similar to the KO localization we previously reported with Ksp-cadherin Cre Bmal1 KO mice, which exhibited nearly complete KO of BMAL1 in the thick ascending limb and collecting duct and partial KO in the distal convoluted tubule (22).

Figure 1.

Knockout (KO) of PERIOD1 (Per1) by Ksp-cadherin Cre is specific to the kidney. A: location of the primers used in recombination PCR to measure the specificity of Per1 KO by Ksp-cadherin Cre. The floxed Per1 schematic shows the placement of loxP sites flanking exons 2–8. The solid black arrow indicates the forward (Fwd) primer directly upstream of the 5′ loxP site. The dashed arrow indicates the loxP reverse (Rev) primer directly downstream of the loxP site in both intact Per1 genes and recombined Per1 genes. PCR using Fwd and loxP primers results in a 705-bp product. The dotted arrow indicates the recomb Rev primer within exon 9. PCR using Fwd and recomb Rev primers results in a 1,318-bp product only in tissues where Ksp-cadherin Cre catalyzes recombination between the two loxP sites, resulting in the deletion of exons 2–8. The PCR product of Fwd and recomb Rev primers in DNA with full-length Per1 is too long to detect under these PCR conditions. B: PCR was performed on the inner medulla, adrenal, and brain genomic DNA of control (CNTL) and kidney-specific Per1 KO (KS-Per1 KO) mice to confirm that recombination is only detected in the kidney of KS-Per1 KO mice. C, CNTL mice; K, KS-Per1 KO mice; loxP, PCR with Fwd and loxP Rev primers; Recomb, PCR with Fwd and recomb Rev primers. Nonspecific bands are present in the loxP reactions. Images are representative of n = 3–6 male mice/group. C: representative RNAscope images of inner medulla sections from kidneys of male CNTL or KS-Per1 KO mice. The black arrows highlight representative punctate, which corresponds to Per1 mRNA. Images were taken at ×40 magnification. Scale bar = 0.05 mm. D: quantification of punctate per mm² for the inner medulla was performed using ImageJ. Values are means ± SE. Genotype effect was determined by Student’s t test. n = 6 mice/group. **P < 0.01.

KS-Per1 KO Mice Maintain Rhythms of Na⁺ Excretion but Have a Higher Na⁺ Balance

Previously, we demonstrated that in response to a HS + DOCP challenge, male global Per1 KO mice had a reduced night-to-day ratio of Na⁺ excretion (12). To determine if male KS-Per1 KO mice had altered rhythms of Na⁺ excretion, male control and KS-Per1 KO mice underwent the same metabolic cage procedures as previously performed with global Per1 KO mice. There was no significant difference in 24-h body weight, average 24-h food intake, or 12-h and average 24-h urine output (Fig. 2, A–E). A significant genotype × treatment interaction was observed in 12-h food intake between KS-Per1 KO and control mice, but post hoc analysis did not indicate any significant individual comparisons.

Figure 2.

Body weight, food intake, and urine output throughout metabolic cage experiments. Male control (CNTL; orange) and Ksp-cadherin Cre kidney-specific PERIOD1 knockout (KS-Per1 KO; blue) C57BL/6 mice (2–3 mo old) were placed in metabolic cages. After 3 days of acclimation on a normal-salt diet (NS), body weight (A), food intake (B), and urine output (D) were measured. Food intake and urine output were measured every 12 h throughout the experiment. The 12-h collections represent the mouse inactive period [day (D) = 6 AM–6 PM] and active period [night (N) = 6 PM–6 AM]. C and E: average (Avg) 24-h food intake (in g; C) and urine output (in g; E) were calculated for each treatment. Values are means   ± SE. Genotype, treatment, and genotype × treatment interaction effects were determined by two-way ANOVA with repeated measures (significant post hoc treatment comparisons are indicated by * on graphs). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 6 male mice/group. HS, high-salt diet; HS + DOCP, high-salt diet plus desoxycorticosterone pivalate treatment.

Urine was collected every 12 h from mice on a NS diet, HS diet, and after DOCP treatment for 3 days, so any differences in urinary excretion represent an acute response to DOCP treatment. There was a significant genotype effect of Na⁺ excretion with KS-Per1 KO mice excreting less Na⁺ than control mice (P_genotype = 0.03; Fig. 3A). Interestingly, there was no significant difference in the night/day pattern of Na⁺ excretion between KS-Per1 KO and control mice throughout the entire experiment. Since there were slight differences between KS-Per1 KO and control mice in food intake, we calculated the Na⁺ balance (Na⁺ intake − urinary Na⁺ output) to determine how much Na⁺ KS-Per1 KO mice were retaining during treatment. KS-Per1 KO mice had a more positive Na⁺ balance and an increased cumulative Na⁺ balance following HS and HS + DOCP treatment compared with control mice (Fig. 3, B and C). There was a significant increase of KS-Per1 KO serum Na⁺ levels in response to HS + DOCP, but this treatment effect was not observed in control mice (Table 1). Serum Na⁺ values fell within range of what has been previously reported in male C57BL/6J mice (12, 23, 24). KS-Per1 KO mice also exhibited a significant increase in mean arterial pressure in response to HS + DOCP, with no significant change in control mice (Fig. 4).

Figure 3.

Male kidney-specific PERIOD1 knockout (KS-Per1 KO) mice have increased Na⁺ balance with high-salt diet (HS) and high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP). Urine Na⁺ was measured using flame photometry. A: total Na⁺ excretion per 12 h revealed significant genotype differences, but no differences were observed in the night (N)-to-day (D) ratio of Na⁺ excretion. B: average (Avg) 24-h Na⁺ balance (intake-output) for each treatment was calculated. C: cumulative Na⁺ balance over the course of the entire experiment was calculated. Values are means ± SE. Genotype, treatment, and genotype × treatment interaction effects were determined by two-way ANOVA with repeated measures (significant post hoc genotype and/or treatment comparisons are indicated by * on graphs; cumulative Na⁺ balance * are significant genotype post hoc comparisons). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 6 male mice/group. BW, body weight; CNTL, control; NS, normal-salt diet.

Table 1.

CNTL and KS-Per1 KO serum Na⁺ and K⁺ on NS and HS + DOCP treatments

	NS	HS + DOCP
CNTL serum Na+	158.6 ± 1.0	158.1 ± 2.9
KS-Per1 KO serum Na+	158.2 ± 1.7	163.8 ± 1.7*
CNTL serum K+	3.6 ± 0.1	2.6 ± 0.2†
KS-Per1 KO serum K+	3.6 ± 0.03	2.7 ± 0.2‡

Values are means ± SE; n = 5–6 male mice/group. Serum concentrations of Na⁺ and K⁺ were determined from samples taken at noon from control (CNTL) and kidney-specific PERIOD1 knockout (KS-Per1 KO) mice on either normal-salt diet (NS) or high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP) treatment. Treatment effects were determined by Student’s t test. *P < 0.05, †P < 0.01, ‡P < 0.001.

Figure 4.

Kidney-specific PERIOD1 knockout (KS-Per1 KO) mice increase blood pressure (BP) in response to high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP). For terminal BP measurements, mice were placed under continuous isoflurane anesthesia with heat support. The telemeter cannula of a PAC10 radiotelemeter was placed in the left carotid artery with the tip of the cannula in the free-flowing blood of the aortic arch. BP was measured on normal-salt diet (NS), high-salt diet (HS), and day 3 HS + DOCP around noon. Genotype, treatment, and genotype × treatment interaction effects were determined by two-way ANOVA (significant post hoc treatment comparisons are indicated by *). *P < 0.05, **P < 0.01. n = 6–8 mice/group. CNTL, control.

We have previously reported that male KS-Per1 KO mice have increased expression of Slc12a3 (NCC) and Scnn1a (αENaC) at baseline compared with control mice (12). To determine whether the genes regulating these transporters were altered in KS-Per1 KO mice, we measured the gene expression of FXYD domain containing ion transport regulator 5 (Fxyd5), GR (Nr3c1), MR (Nr3c2), serum/glucocorticoid-regulated kinase 1 (Sgk1), and nitric oxide synthase 1 (Nos1) following either NS diet or HS + DOCP treatment in KS-Per1 KO and control mice. Fxyd5 expression significantly increased in the KS-Per1 KO renal cortex in response to HS + DOCP treatment (P_treatment = 0.001; Fig. 5A). Renal cortex expression of Nr3c1 and Nr3c2 significantly decreased following HS + DOCP treatment in control mice (∼40% reduction), but this treatment response was not significant in KS-Per1 KO mice (interaction P_{genotype × treatment} < 0.05; Fig. 5, B and C). Control mice significantly increased Sgk1 renal cortex expression after DOCP administration (P_treatment = 0.0005), but Sgk1 expression did not significantly change in KS-Per1 KO mice after HS + DOCP (P_genotype = 0.03; Fig. 5D). Renal medulla Nos1 gene expression analysis revealed a significant interaction between genotype and treatment (P_{genotype × treatment} = 0.04; Fig. 5E).

Figure 5.

Gene expression in the kidneys of control (CNTL) and kidney-specific PERIOD1 knockout (KS-Per1 KO) mice on a normal-salt diet (NS) and with high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP) treatment. Kidneys from CNTL (orange) and KS-Per1 KO (blue) mice on NS or HS + DOCP were collected at noon. The kidneys were dissected into the cortex (Ctx) and whole medulla (Med) before RNA isolation and subsequent cDNA conversion. Quantitative PCR was performed to evaluate various Na⁺-handling genes. Expression was relativized to CNTL normal salt values. A–D: relative expression of FXYD domain containing ion transport regulator 5 (Fxyd5; A), glucocorticoid receptor (Nr3c1; B), mineralocorticoid receptor (Nr3c2; C), and serum/glucocorticoid-regulated kinase 1 (Sgk1; D) in the kidney cortex. E: relative expression of nitric oxide synthase 1 (Nos1) in the kidney whole medulla. Values are means ± SE. Genotype, treatment, and genotype × treatment interaction effects were determined by two-way ANOVA (significant post hoc genotype and/or treatment comparisons are indicated by *). *P < 0.05, **P < 0.01, ***P < 0.001. n = 5–6 male mice/group. CNTL, control.

KS-Per1 KO Mice Have Higher Aldosterone and ET-1 Levels

Previously, we reported that male global Per1 KO mice exhibited increased aldosterone and ET-1 levels (19). Aldosterone regulates Na⁺ handling, and since KS-Per1 KO mice seem to be more sensitive to dietary Na⁺ than control mice, serum aldosterone levels were measured. KS-Per1 KO serum aldosterone was significantly higher compared with control mice on the NS diet (20.6 vs. 11.4 pg/mL/g body wt, P_genotype = 0.004; Fig. 6A). Both KS-Per1 KO and control mice suppressed aldosterone levels in response to HS + DOCP treatment (P_treatment < 0.0001). ET-1 peptide also regulates Na⁺ handling through its actions on ET type A and type B (ET_A and ET_B, respectively) receptors. KS-Per1 KO mice had increased urinary ET-1 and medulla ET-1 peptide levels after HS + DOCP compared with control mice (Fig. 6, B and C).

Figure 6.

Male kidney-specific PERIOD1 knockout (KS-Per1 KO) mice have increased serum aldosterone and production of endothelin-1 (ET-1). A: blood was collected from control (CNTL; orange) mice and KS-Per1 KO (blue) mice on normal-salt diet (NS) and high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP) treatments at noon. Serum aldosterone was measured by ELISA. Urinary ET-1 (B) or medullary peptide ET-1 (C) on HS + DOCP day 4 was measured by ELISA. Values are means ± SE. Genotype, treatment, and genotype × treatment interaction effects were determined by two-way ANOVA (significant post hoc genotype and/or treatment comparisons are indicated by *). Genotype effects on HS + DOCP medullary ET-1 peptide levels were determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 6–7 mice/group. Avg, average; BW, body weight.

KS-Per1 KO Mice Have Altered Adrenal Gland Gene Expression

As KS-Per1 KO mice have higher serum aldosterone than control mice, adrenal gland gene expression was measured to determine if KO of Per1 in the kidney resulted in transcriptional changes that could result in increased aldosterone production. Indeed, Cyp11b2, which encodes aldosterone synthase, the enzyme responsible for the synthesis of aldosterone, was significantly increased in KS-Per1 KO mice compared with control mice on the NS diet (∼40% increase, P_genotype = 0.02; Fig. 7A). Expression of Cyp11b2 was significantly suppressed in both KO and control mice following HS + DOCP treatment, mimicking the treatment effect on serum aldosterone (Fig. 6). However, there was no significant genotype or treatment effect on the expression of 3β-hydroxysteroid dehydrogenase (Hsd3b6), a key regulator of aldosterone biosynthesis that converts pregnenolone to progesterone in mice (Fig. 7B).

Figure 7.

Altered gene expression in the adrenals of kidney-specific PERIOD1 knockout (KS-Per1 KO) mice. KS-Per1 KO (blue) and control (CNTL; orange) adrenals were collected at noon on a normal-salt diet (NS) and high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP) treatment. RNA was isolated and converted to cDNA using random hexamers. Relative gene expression was measured using a TaqMan assay with β-actin as the reference gene. Expression was relativized to CNTL NS values. A and B: relative expression of the aldosterone synthase gene (Cyp11b2; A) and the 3β-hydroxysteroid dehydrogenase gene (Hsd3b6; B) were measured. C–G: relative expression of the circadian clock genes Reverbα (C), Bmal1 (D), Clock (E), Cry1 (F), and Cry2 (G) was also measured. Values are means ± SE. Genotype, treatment, and genotype × treatment interaction effects were determined by two-way ANOVA (significant post hoc genotype and/or treatment comparisons are indicated by *). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 5 or 6 male mice/group.

Next, the expression of adrenal clock genes was assessed. Expression of Reverbα, which encodes a negative regulator of BMAL1, was significantly increased in both KS-Per1 KO and control mice following HS + DOCP treatment (∼50% increase, P_treatment < 0.0001; Fig. 7C). No genotype or treatment effects were seen in Bmal1 or Clock expression (Fig. 7, D and E). Expression of Cry1 significantly decreased in KS-Per1 KO mice in response to HS + DOCP treatment (∼25% decrease, P_treatment < 0.0001; Fig. 7F). Interestingly, there was a significant genotype effect to increase Cry2 expression (P_genotype = 0.008; Fig. 7G).

DISCUSSION

The main findings of this study are that KO of PER1 in the distal nephron and collecting duct results in increased aldosterone levels and increased renal Na⁺ retention in response to HS + DOCP treatment. These effects are associated with changes in expression of several renal Na⁺-handling genes as well as changes in gene expression in the adrenal gland. Overall, these data suggest an important role for PER1 in the kidney in regulating kidney/adrenal cross talk and maintaining homeostasis.

Previously, we established that male global Per1 KO mice have reduced night/day rhythms of Na⁺ excretion following HS + DOCP treatment (12). In this study, we demonstrated that renal Na⁺ excretion rhythms in male KS-Per1 KO mice remain intact, even after HS + DOCP treatment, suggesting that distal nephron PER1 is not required to maintain night/day differences in Na⁺ excretory rhythm. This raises the question of the mechanisms behind blunted night/day Na⁺ excretory rhythms in response to HS + DOCP in global Per1 KO mice. Tissue-specific KO mouse lines using Ksp-cadherin Cre recombinase result in gene KO within the renal distal nephron and collecting duct (22). Therefore, PER1 in other segments of the nephron could be involved in the regulation of renal Na⁺-handling rhythms. However, KS-Per1 KO mice display increased Na⁺ retention when challenged with HS diet or HS + DOCP treatment, supporting the hypothesis that the circadian clock transcription factor PER1 in the distal nephron and collecting duct of the kidney plays a key role in the regulation of renal Na⁺ handling that may be independent of time of day.

We have previously published preliminary gene expression data showing that NCC and αENaC were inappropriately upregulated in KS-Per1 KO mice compared with control mice at baseline, further suggesting that distal nephron PER1 regulates renal Na⁺ handling (12). In an effort to delineate the mechanisms behind the upregulation of these transporters and the increased Na⁺ retention phenotype in KS-Per1 KO mice, we assessed the expression of genes known to encode proteins that regulate renal Na⁺ handling. FXYD5 interacts with Na⁺-K⁺-ATPase, increasing its activity (25). There was no significant difference between control and KS-Per1 KO Fxyd5 expression (Fig. 5A). However, Fxyd5 was increased in both control and KS-Per1 KO mice following HS + DOCP, which could be a mechanism to increase K⁺ reabsorption and limit further Na⁺ reabsorption along the nephron, as both KS-Per1 KO and control mice had low serum K⁺ levels (Table 1). The increased Nos1 expression in KS-Per1 KO mice following HS + DOCP could be a potential compensatory mechanism to limit Na⁺ reabsorption via ENaC, as NOS1 has been shown to be a negative regulator of ENaC activity (26). Renal GR activation has been shown to impact Na⁺ transport along the length of the renal tubule by activating Na⁺ transporters, including NCC (27). In addition, GR activation has been shown to stimulate ENaC in vitro (28) and possibly in vivo (29). Although control mice have significantly reduced expression of renal GR mRNA following HS + DOCP, there was no reduction in GR expression in KS-Per1 KO mice (Fig. 5B). MR mRNA renal expression was significantly reduced following HS + DOCP treatment in control mice, but there was no change in expression in KS-Per1 KO mice (Fig. 5C). MR activation promotes transcription of Sgk1, and SGK1 increases the activity of ENaC (30, 31). In addition, renal expression of Sgk1 was significantly increased in KS-Per1 KO mice compared with control mice (Fig. 5D). It is tempting to speculate that altered expression of MR, GR, and SGK1 in KS-Per1 KO mice could be a potential mechanism for the observed increased Na⁺ retention. Future studies will be directed toward testing the functional significance of PER1-mediated regulation of these genes related to the regulation of renal Na⁺ handling. Consistent with our overall hypothesis, KS-Per1 KO mice have a significant increase in BP in response to HS + DOCP, whereas control mice do not. A limitation of these results is that it is a snapshot measurement. Future studies will look to measure BP and the circadian BP profile using gold-standard radiotelemetry in KS-Per1 KO and control mice in response to HS + DOCP. Our laboratory was the first to identify Per1 and the ET-1 gene, Edn1, as aldosterone target genes in mouse inner medullary collecting duct cells (11). In addition, Per1 transcription has also been shown to be stimulated by aldosterone in cardiomyocytes through activation of MR signaling (32). Renal MR and Sgk1 are also aldosterone-responsive genes; MR and SGK1 proteins are involved in the activation of NCC and αENaC (30, 38). Similar to the increased urinary aldosterone previously observed in male global Per1 KO mice (19), KS-Per1 KO mice exhibited higher levels of serum aldosterone on a NS diet (Fig. 6A). Importantly, both male global Per1 KO mice and KS-Per1 KO mice suppressed aldosterone levels in response to HS + DOCP.

Consistent with increased aldosterone, adrenal gland mRNA expression of Cyp11b2, which encodes aldosterone synthase, was upregulated in KS-Per1 KO mice compared with control mice (Fig. 7A). This further highlights a relationship between PER1 and aldosterone and the importance of PER1 in proper cross talk between the kidney and adrenal gland to maintain homeostasis. Synthesis of aldosterone is predominately under the control of the renin-angiotensin-aldosterone system, K⁺, and, to a lesser extent, the hypothalamic-pituitary-adrenal axis (33). Whether PER1 in the kidney communicates with these systems to regulate aldosterone or impact negative feedback of these systems is not fully understood and remains a critical gap in our knowledge of the role of the kidney clock in integrative physiology. ET-1 can also regulate renal Na⁺ handling through its actions on ET_A and ET_B receptors (34, 35). KS-Per1 KO mice exhibited increased urinary ET-1 excretion, similar to global Per1 KO mice (19), as well as higher medullary ET-1 peptide levels (Fig. 6, B and C). These data, along with our previously reported work, suggest that PER1 in the kidney acts as a negative regulator of ET-1. Increased ET-1 is associated with increased aldosterone in mice (36), and Edn1 gene variants are associated with dysregulation aldosterone secretion in humans (37). Thus, it is tempting to speculate that regulation of ET-1 may be one mechanism by which PER1 contributes to kidney/adrenal cross talk. Further work will be needed to determine the mechanism of renal PER1 in the regulation of aldosterone and ET-1 and its relationship to the increased Na⁺ retention in KS-Per1 KO mice.

Although there is an increasing number of published studies examining the effect of tissue-specific clock gene KO on physiological functions, there is extremely limited knowledge regarding KO of a circadian clock gene in one tissue and its impacts on gene expression in other tissues. Previous studies have shown that peripheral clocks communicate with each other through the release of hormones, such as glucocorticoids, insulin, and glucagon, in addition to autonomic nervous system signaling (40). However, it is not clear what impact disruption of one peripheral clock gene has on the other peripheral clocks in terms of gene regulation. Here, we report the novel finding that renal-specific KO of the circadian clock gene Per1 results in alterations of adrenal gene expression. The clock gene Cry2 was increased in KS-Per1 KO adrenals compared with control adrenals with both NS diet and HS + DOCP treatment. The role of Cry2 in the adrenal gland remains unclear. Previous work investigating the effect of in vitro knockdown of CRY2 in adrenal explants of capuchin monkeys suggested a role for adrenal CRY2 in the regulation of steroidogenesis (39). However, this study examined specific effects on adrenocorticotropic hormone stimulation and cortisol production; therefore, further work is needed to investigate the role of CRY2 in regulating aldosterone synthesis. It is interesting to note that global double KO of Cry1 and Cry2 resulted in increased aldosterone levels (41). Peripheral clock cross talk may contribute to the regulation of renal and adrenal function to maintain homeostasis.

Given the PER1-dependent renal Na⁺ retention phenotype and because PER1 is a transcription factor, one focus of this study was to investigate the effect of Per1 KO on the transcription of renal Na⁺-handling genes and adrenal genes involved in steroidogenesis. One limitation of this study is that protein expression was not assessed. In addition, gene expression was only measured at one time point, noon, the middle of the mouse inactive period. We focused on this time point because of our previous data with global Per1 KO mice, which showed that renal Na⁺-handling genes were dysregulated at this time, along with changes in Na⁺ excretion patterns (12). The PER1-dependent renal Na⁺ retention phenotype was not in conjunction with increased body mass. Na⁺ balance was only measured in terms of intake and urinary output. Na⁺ can also be lost via the skin or feces, so future studies should assess this to determine overall Na⁺ balance. Furthermore, mild hyperaldosteronism leads to increased plasma Na⁺ due to resetting of the osmostat and not excess Na⁺ retention (42). Future studies will look to measure serum copeptin levels and osmolality to determine its role in increased serum Na⁺ in KS-Per1 KO mice. Another limitation is that this study was only performed in male mice. This study focused on male mice because we have previously reported that, unlike male global Per1 KO mice, female global Per1 KO mice did not exhibit altered night/day rhythms of Na⁺ excretion following HS + DOCP treatment (19). Furthermore, there were no changes in serum Na⁺, αENaC gene expression, or serum aldosterone concentration in female Per1 KO mice (43). Future studies are needed to determine the mechanism of these sex differences in the regulation of cardiovascular function by PER1.

Perspectives and Significance

Here, we show, for the first time, that KS-Per1 KO male mice exhibit increased renal Na⁺ retention, yet maintain normal diurnal changes in Na⁺ excretion, unlike global Per1 KO mice. In response to HS + DOCP treatment, KS-Per1 KO mice do not decrease renal GR and MR transcription as control mice do, which may contribute to the increased Na⁺ balance observed in these mice. These results support the idea that the circadian clock is not only important in maintaining rhythms of physiological functions but is also important for adaptability in response to environmental cues, such as a HS diet, to maintain overall homeostasis. In addition, KS-Per1 KO mice have increased serum aldosterone and altered adrenal gland gene expression compared with control mice. These data suggest that PER1 is required for proper cross talk between the kidney and adrenal gland to regulate aldosterone levels.

SUPPLEMENTAL DATA

Supplemental Figs. S1 and S2: https://doi.org/10.6084/m9.figshare.18416846.v1.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01DK109570 and R03DK098460 (to M.L.G.) and T32DK104721 (to L.G.D.), American Heart Association (AHA) Grant-In-Aid 16GRNT31220009 (to M.L.G.), an AHA Established Investigator Award (to M.L.G.), and AHA Postdoctoral Fellowship 18POST34030210 (to L.G.D.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.G.D., H.M.C., C.S.W., and M.L.G. conceived and designed research; L.G.D., H.M.C., G.R.C., K-Y.C., I.J.L., A.J., D.B., S.M., E.R., K.B., W.L., P.B., and M.L.G. performed experiments; L.G.D., H.M.C., G.R.C., K-Y.C., I.J.L., A.J., D.B., S.M., E.R., K.B., W.L., P.B., and M.L.G. analyzed data; L.G.D., H.M.C., G.R.C., I.J.L., A.J., D.B., S.M., E.R., K.B., W.L., P.B., C.S.W., and M.L.G. interpreted results of experiments; L.G.D., H.M.C., G.R.C., K-Y.C., D.B., and P.B. prepared figures; L.G.D., H.M.C., and M.L.G. drafted manuscript; L.G.D., H.M.C., G.R.C., A.J., C.S.W., and M.L.G. edited and revised manuscript; L.G.D., H.M.C., G.R.C., K-Y.C., I.J.L., A.J., D.B., S.M., E.R., K.B., W.L., P.B., C.S.W., and M.L.G. approved final version of manuscript.