Loss of the liver circadian clock affects the expression of intrarenal renin-angiotensin system components

Fig. 1. Effect of targeted deletion of hepatic circadian clock genes on liver sysRAAS components. Relative expression of (a) Agt, (b) Enpep, (c) Lnpep, (d) Bmal1, (e) Per1, and (f) Per2 in LCre (black solid lines), LBmal1 (green dotted lines) and LPer1/2 (magenta dashed lines) mice at six different zeitgeber time points with 4-hour intervals. Green stars indicate significant differences between LCre and LBmal1, and magenta stars indicate significant differences between LCre and LPer1/2. Data are presented as mean ± SEM (n = 3). (g) western blot analysis of the indicated proteins at ZT8 and quantification (n = 4) (h-j). On the left are the positions of size markers [kDa]. Agt, AGT: Angiotensinogen; Enpep: Glutamyl aminopeptidase; Lnpep: Leucyl and Cystinyl aminopeptidase; Bmal1, BMAL1: Brain and muscle ARNT-like protein 1; Per1: Period 1; Per2, PER2: Period 2; HSP90: heat shock protein 90. Two-way ANOVA with Tukey’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 2. Effect of targeted deletion of hepatic circadian clock genes on irRAS components and renal clock genes. Relative expression of (a) Agt, (b) Ace, (c) Ren1, (d) Nhe3, (e) Agtr1, (f) Nr1h3, (g) Bmal1, (h) Per1 and (i) Per2 in LCre (black solid lines), LBmal1 (green dotted lines) and LPer1/2 (magenta dashed lines) mice at six different zeitgeber time points with 4-hour intervals. Green stars indicate significant differences between LCre and LBmal1, and magenta stars indicate significant differences between LCre and LPer1/2. Data are presented as mean ± SEM (n = 3). Agt: Angiotensinogen; Ace: Angiotensin I-converting enzyme; Ren1: Renin 1; Nhe3: Na+/H+ exchanger 3; Agtr1: Angiotensin II receptor type 1; Nr1h3: Liver X receptor α; Bmal1: Brain and muscle ARNT-like protein 1; Per1: Period 1; Per2: Period 2. Two-way ANOVA with Tukey’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 3. Analysis of the median phase differences for kidney genes in the different genotypes. Left side: Plot of the calculated phases from Supplemental table S1 on a 24 h circle for LCre (black dots) and LBmal1 (green squares) mice. Right side: Plot of the calculated phases from Supplemental table S1 on a 24 h circle for LCre (black dots) and LPer1/2 (magenta triangles) mice. Bottom: calculation of the median phase of gene expression ± circular standard deviation in hours; Watson’s two sample test of homogeneity comparing LCre with LBmal1, or LCre with LPer1/2.

Fig. 4. Effect of targeted deletion of hepatic circadian clock genes on plasma sysRAAS components. (a) AGT, (b) renin, (c) ACE, and (d) aldosterone in LCre (black solid lines), LBmal1 (green dotted lines) and LPer1/2 (magenta dashed lines) mice at six different zeitgeber time points with 4-hour intervals. Green stars indicate significant differences between LCre and LBmal1, and magenta stars indicate significant differences between LCre and LPer1/2. Data are presented as mean ± SEM (n = 3). AGT: Angiotensinogen. ACE: Angiotensin I-converting enzyme. Two-way ANOVA with Tukey’s post hoc test, * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 5. Effect of targeted deletion of hepatic circadian clock genes on blood pressure and peptide hormone accumulation at ZT8. (a) systolic and (b) diastolic blood pressure of LCre, LBmal1, and LPer1/2 mice. (c) concentration of AGT in plasma, (d) Ang I, (e) Ang II, (f) Ang III, (g) Ang IV. (h) renin activity compared to standard. AGT: angiotensinogen; Ang: angiotensin. Data are presented as mean ± SEM (n = 6). One-way ANOVA with Dunnett’s post hoc test compared to LCre, ns: non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 6. Model. Comparison of two different tissues in two different mouse strains. In wild type mice (yellow box), liver and kidney have a functional circadian oscillator: Per1 and Per2 expression peak in the light phase (magenta), while Bmal1 expression peaks in the dark phase (green). The systemic RAAS (orange) and intrarenal RAS (blue) components are synchronized and peak during the dark phase. In liver-specific knock-out mice (light blue box) there is no functional oscillator in the liver, while the circadian oscillator in the kidney remains unaffected. Surprisingly, the systemic RAAS and the intrarenal RAS components are still partially synchronized albeit their peak is shifted towards the light phase.