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. 2014 Jul;25(7):1430-9.
doi: 10.1681/ASN.2013060641. Epub 2014 Mar 20.

Local renal circadian clocks control fluid-electrolyte homeostasis and BP

Natsuko Tokonami  1 , David Mordasini  1 , Sylvain Pradervand  2 , Gabriel Centeno  1 , Céline Jouffe  3 , Marc Maillard  4 , Olivier Bonny  5 , Frédéric Gachon  3 , R Ariel Gomez  6 , Maria Luisa S Sequeira-Lopez  6 , Dmitri Firsov  7
Affiliations

Local renal circadian clocks control fluid-electrolyte homeostasis and BP

Natsuko Tokonami et al. J Am Soc Nephrol. 2014 Jul.

Abstract

The circadian timing system is critically involved in the maintenance of fluid and electrolyte balance and BP control. However, the role of peripheral circadian clocks in these homeostatic mechanisms remains unknown. We addressed this question in a mouse model carrying a conditional allele of the circadian clock gene Bmal1 and expressing Cre recombinase under the endogenous Renin promoter (Bmal1(lox/lox)/Ren1(d)Cre mice). Analysis of Bmal1(lox/lox)/Ren1(d)Cre mice showed that the floxed Bmal1 allele was excised in the kidney. In the kidney, BMAL1 protein expression was absent in the renin-secreting granular cells of the juxtaglomerular apparatus and the collecting duct. A partial reduction of BMAL1 expression was observed in the medullary thick ascending limb. Functional analyses showed that Bmal1(lox/lox)/Ren1(d)Cre mice exhibited multiple abnormalities, including increased urine volume, changes in the circadian rhythm of urinary sodium excretion, increased GFR, and significantly reduced plasma aldosterone levels. These changes were accompanied by a reduction in BP. These results show that local renal circadian clocks control body fluid and BP homeostasis.

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Figures

Figure 1.
Figure 1.
Ren1dCre-mediated Bmal1 inactivation is limited to the kidney. (A) qPCR analysis of Cre mRNA expression (ZT0) in various tissues of control (white bars) and Bmal1lox/lox/Ren1dCre (black bars) mice (primer references are in Supplemental Material); data are mean±SEM (n=5). (B) RT-PCR analysis of Bmal1 mRNA expression (ZT0) in different tissues of Bmal1lox/lox/Ren1dCre mice performed with primers flanking the floxed region. mRNA extracted from kidneys of whole-body (total) knockout (KO) of Bmal1 was used as a control (primer sequences are in Supplemental Material). WT, wild type. (C) qPCR analysis of Bmal1 mRNA expression (ZT0) in various tissues of control (white bars) and Bmal1lox/lox/Ren1dCre (black bars) mice (primer references are in Supplemental Material). Data are mean±SEM (n=5). *P<0.05, t test. Tissues were collected from mice euthanized at ZT0.
Figure 2.
Figure 2.
The Ren1dCre mice express the Cre recombinase in the juxtoglomerular apparatus and in the collecting duct. Immunohistochemical localization of Cre recombinase in the renal cortex (ZT4). Strong nuclear Cre staining (black) is present in the arterioles of the juxtaglomerular apparatus (red arrows) and the collecting duct (blue arrows), which is evidenced by costaining with aquaporin-2 (AQP2) water channel (red).
Figure 3.
Figure 3.
The Bmal1 is inactivated in multiple tubular segments in kidneys of Ren1dCre mice. (A) mRNA and (B) protein expression of Bmal1 in microdissected glomeruli (glom), proximal convoluted tubule (PCT), proximal straight tubule (PST), MTAL, cortical thick ascending limb (CTAL), distal convoluted tubule (DCT), connecting tubule (CNT), CCD, and OMCD. Microdissection was performed at ZT4. (B) Whole-cell lysates of 20-mm microdissected nephron segments were used for Western blotting (n=3). (A) Data are mean±SEM (n=3). **P<0.01; ***P<0.005, t test. Tissues were collected from mice euthanized at ZT4. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 4.
Figure 4.
Circadian patterns of renin mRNA and protein expression are significantly modified in kidneys of Ren1dCre mice. Circadian patterns of renin mRNA and protein expression in control and Bmal1lox/lox/Ren1dCre mice. (A) Circadian pattern of renin mRNA expression in kidneys of control (black line) and Bmal1lox/lox/Ren1dCre (red line) mice (n=6/time point). Kidneys were extracted from mice euthanized at indicated circadian time points. Data are mean±SEM (n=6). Significant ANOVA factors: Pgenotype=0.003, Ptime=0.01. (B) Circadian pattern of renin protein expression in kidneys of control mice. This representative Western blot was performed on samples prepared by combining equivalent amounts of protein extracted from kidneys of six independent mice in each time point. (C) Circadian pattern of renin protein expression in kidneys of Bmal1lox/lox/Ren1dCre mice. This representative Western blot was performed on samples prepared by combining equivalent amounts of protein extracted from kidneys of six independent mice in each time point. (D) Densitometry analysis of Western blots performed on the individual samples of kidney protein extracts used in B (control mice; black line) and C (Bmal1lox/lox/Ren1dCre mice; red line; n=6/time point). Data are mean±SEM (n=6). Significant ANOVA factors: Pgenotype=0.04.
Figure 5.
Figure 5.
Circadian patterns of PRC, plasma aldosterone levels, and plasma angiotensinogen concentration are significantly modified in Ren1dCre mice. Circadian patterns of (A) PRC, (B) plasma aldosterone level, and (C) plasma angiotensinogen concentration in control (white bars) and Bmal1lox/lox/Ren1dCre (black bars) mice (n=10–12/time point). Data are mean±SEM (n=10–12 depending on time point); t test significance: *P<0.05; **P<0.01; ***P<0.005. Significant ANOVA factors: (A) Ptime<0.001; (B) Pgenotype<0.001, Ptime<0.001, Pinteraction<0.001; (C) Pgenotype=0.01, Ptime<0.001.
Figure 6.
Figure 6.
Local renal circadian clocks control the dynamics of urinary sodium excretion. Circadian patterns of (A) urinary water, (B) sodium, and (C) potassium excretion and (D) urinary sodium-to-potassium ratios in control (black line) and Bmal1lox/lox/Ren1dCre (red line) mice. (A) Circadian pattern of urinary water excretion was determined on the urine collected hourly over the 24-hour circadian cycle. The dynamics of (B) urinary sodium and (C) potassium excretion and (D) the sodium-to-potassium ratio were determined on the urine collected hourly over the 12 hours of the activity phase of the circadian cycle (ZT12–ZT24). Data are mean±SEM (n=21). Significant ANOVA factors: (B) Ptime<0.001, Pinteraction<0.001; (C) Ptime=0.01; (D) Ptime<0.001, Pinteraction<0.001.
Figure 7.
Figure 7.
Local renal circadian clocks are required for BP control. (A) Systolic and (B) diastolic BP in control (black line) and Bmal1lox/lox/Ren1dCre (red line) mice. For each mouse, a mean of 6-day recordings was calculated (the 6-day recordings are shown in Supplemental Figure 9); then, the mean of these values was determined for each genotype. Data are mean±SEM (n=7 for both genotypes). Significant ANOVA factors: (B) Ptime<0.001, Pgenotype<0.001.
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