Development of the circadian clockwork in the kidney Krisztina Mészáros, Linda Pruess, Attila J. Szabó, Matthias Gondan, Eberhard Ritz, Franz Schaefer  Kidney International  Volume 86, Issue 5, Pages 915-922 (November 2014) DOI: 10.1038/ki.2014.199 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Postnatal development of expression of clock genes Bmal1, Rev-erbα, Per2, and Cry1 in the kidney. Male offspring (n=7) were killed every 4h (ZT2-22; ZT=Zeitgeber time, ZT0 is light onset, at 0600 hours). Expression of clock gene in the kidney was measured at embryonic day 20 (E20, panel a) and at postnatal week 1 (1W, b), 4 (4W, c), and 12 (12W, d). Circadian time effects were analyzed by the single cosinor procedure. The characteristics of the rhythms were described by the peak of the oscillation wave marked with arrows, i.e., the acrophase ϕ (hh:mm) and the double amplitude, 2A (%). The data are listed in Supplementary Table S2. P-values refer to statistical tests of the alternative hypothesis 2A>0, with P<0.05 considered as statistically significant. E20: embryonic day 20; 1W, 4W, 12W: postnatal age 1, 4, and 12 weeks. Kidney International 2014 86, 915-922DOI: (10.1038/ki.2014.199) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Postnatal development of the expression of clock-controlled genes αENaC, SGK1, NHE3, and AVPR2 in the kidney. For methodological details see Figure 1. E20: embryonic day 20 (panel a); 1W (b), 4W (c), 12W (d): postnatal age 1, 4, and 12 weeks. Kidney International 2014 86, 915-922DOI: (10.1038/ki.2014.199) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Circadian gene expression during ontogenesis of the kidney. Clock genes are plotted on the left, clock-controlled genes on the right. Acrophase (ϕ) is the time of cosine wave peak (a). 2A: double amplitude expressed as % of a 24-hour mean (mesor, b). ZT=Zeitgeber time, ZT0 is light onset, at 0600 hours. E20: embryonic day 20; 1W, 4W, 12W: postnatal age 1, 4, and 12 weeks. Kidney International 2014 86, 915-922DOI: (10.1038/ki.2014.199) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Patterns of motion activity, food and water intake, and feeding time, shown as % of daily total. ZT0 is light onset (at 0600 hours). Timing of feeding showed a diurnal pattern. Although the mothers and the offsprings at 4 and 12 weeks of age were active during the dark period, food intake occurred mostly during the night. Triangles indicate acrophases of circadian gene expression. Kidney International 2014 86, 915-922DOI: (10.1038/ki.2014.199) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Circadian renal clock gene expression of 1-week-old pups after 7 days of daily withdrawal of the mother. The pups were separated from their mothers for 4h (ZT3-7, shown by the shaded area) daily. The results for the maternal withdrawal animals are superimposed on the control group who remained with their mother for 24h (1W, dark bars and black lines, statistics in the first line). The white bars and the dashed lines represent the maternal withdrawal pups (MW, statistics in the second line). For methodological details see Figure 1. 1W, 1 week. Kidney International 2014 86, 915-922DOI: (10.1038/ki.2014.199) Copyright © 2014 International Society of Nephrology Terms and Conditions