The circadian clock is disrupted in mice with adenine-induced tubulointerstitial nephropathy

Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide health problem. Sleep disorders are prevalent in patients with CKD raising the possibility that these patients have a disorganized circadian timing system. Here, we examined the effect of adenine-induced tubulointerstitial nephropathy on the circadian system in mice. Compared to controls, adenine-treated mice showed serum biochemistry evidence of CKD as well as increased kidney expression of inflammation and fibrosis markers. Mice with CKD exhibited fragmented sleep behavior and locomotor activity, with lower degrees of cage activity compared to mice without CKD. On a molecular level, mice with CKD exhibited low amplitude rhythms in their central circadian clock as measured by bioluminescence in slices of the suprachiasmatic nucleus of PERIOD 2::LUCIFERASE mice. Whole animal imaging indicated that adenine treated mice also exhibited dampened oscillations in intact kidney, liver, and submandibular gland. Consistently, dampened circadian oscillations were observed in several circadian clock genes and clock-controlled genes in the kidney of the mice with CKD. Finally, mice with a genetically disrupted circadian clock (Clock mutants) were treated with adenine and compared to wild type control mice. The treatment evoked worse kidney damage as indicated by higher deposition of gelatinases (matrix metalloproteinase-2 and 9) and adenine metabolites in the kidney. Adenine also caused non-dipping hypertension and lower heart rate. Thus, our data indicate that central and peripheral circadian clocks are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock accelerates CKD progression. Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide health problem. Sleep disorders are prevalent in patients with CKD raising the possibility that these patients have a disorganized circadian timing system. Here, we examined the effect of adenine-induced tubulointerstitial nephropathy on the circadian system in mice. Compared to controls, adenine-treated mice showed serum biochemistry evidence of CKD as well as increased kidney expression of inflammation and fibrosis markers. Mice with CKD exhibited fragmented sleep behavior and locomotor activity, with lower degrees of cage activity compared to mice without CKD. On a molecular level, mice with CKD exhibited low amplitude rhythms in their central circadian clock as measured by bioluminescence in slices of the suprachiasmatic nucleus of PERIOD 2::LUCIFERASE mice. Whole animal imaging indicated that adenine treated mice also exhibited dampened oscillations in intact kidney, liver, and submandibular gland. Consistently, dampened circadian oscillations were observed in several circadian clock genes and clock-controlled genes in the kidney of the mice with CKD. Finally, mice with a genetically disrupted circadian clock (Clock mutants) were treated with adenine and compared to wild type control mice. The treatment evoked worse kidney damage as indicated by higher deposition of gelatinases (matrix metalloproteinase-2 and 9) and adenine metabolites in the kidney. Adenine also caused non-dipping hypertension and lower heart rate. Thus, our data indicate that central and peripheral circadian clocks are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock accelerates CKD progression. Translational StatementSleep disorders are prevalent in chronic kidney disease patients, although the underlying mechanisms are not understood. The current study demonstrates that adenine-induced tubulointerstitial nephropathy disrupted the circadian system both centrally and in peripheral organs. Clock mutant mice were also more vulnerable to the effects of adenine. These findings aid the understanding of sleep disturbances in adenine phosphoribosyltransferase deficiency, a rare inherited metabolic disorder that leads to the accumulation of 2,8-dihydroxyadenine. More broadly, the results suggest that circadian disruption caused by environmental factors such as nighttime shift work may be a risk factor for chronic kidney disease development. Sleep disorders are prevalent in chronic kidney disease patients, although the underlying mechanisms are not understood. The current study demonstrates that adenine-induced tubulointerstitial nephropathy disrupted the circadian system both centrally and in peripheral organs. Clock mutant mice were also more vulnerable to the effects of adenine. These findings aid the understanding of sleep disturbances in adenine phosphoribosyltransferase deficiency, a rare inherited metabolic disorder that leads to the accumulation of 2,8-dihydroxyadenine. More broadly, the results suggest that circadian disruption caused by environmental factors such as nighttime shift work may be a risk factor for chronic kidney disease development. The number of dialysis patients has been increasing around the world, and chronic kidney disease (CKD) has become a global health issue.1Lysaght M.J. Maintenance dialysis population dynamics: current trends and long-term implications.J Am Soc Nephrol. 2002; 13: S37-S40Crossref PubMed Google Scholar Currently, there is no cure for CKD, and treatment options, including kidney transplantation or a lifetime of dialysis, are limited. Further, CKD patients have a high risk of complications, including stroke,2El Husseini N. Kaskar O. Goldstein L.B. Chronic kidney disease and stroke.Adv Chron Kidney Dis. 2014; 21: 500-508Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar cardiovascular disease,3Foley R.N. Clinical epidemiology of cardiovascular disease in chronic kidney disease.J Renal Care. 2010; 36: 4-8Crossref PubMed Scopus (67) Google Scholar fractures,4Alem A.M. Sherrard D.J. Gillen D.L. et al.Increased risk of hip fracture among patients with end-stage renal disease.Kidney Int. 2000; 58: 396-399Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar and sarcopenia.5Sato E. Mori T. Mishima E. et al.Metabolic alterations by indoxyl sulfate in skeletal muscle induce uremic sarcopenia in chronic kidney disease.Sci Rep. 2016; 6: 36618Crossref PubMed Scopus (121) Google Scholar Therefore, it is important to investigate approaches to reduce the risk of CKD early in the disease progression. Several studies have reported that CKD patients show fragmentation of sleep, shorter sleep duration, poor sleep quality, and difficulties in the timing of sleep.6Agarwal R. Light R.P. Sleep and activity in chronic kidney disease: a longitudinal study.Clin J Am Soc Nephrol. 2011; 6: 1258-1265Crossref PubMed Scopus (58) Google Scholar More than 50% of CKD patients suffer from daytime sleepiness.7Hanly P. Sleep apnea and daytime sleepiness in end-stage renal disease.Semin Dial. 2004; 17: 109-114Crossref PubMed Scopus (101) Google Scholar Clinical reports show abnormal electroencephalogram patterns in CKD patients during wakefulness, especially at the later stages of the disease,8Gadewar P. Acharya S. Khairkar P. et al.Dynamics of electroencephalogram (EEG) in different stages of chronic kidney disease.J Clin Diagn Res. 2015; 9: OC25-OC27PubMed Google Scholar and a positive correlation between the abnormal electroencephalogram and serum (blood) urea nitrogen (BUN) has been reported.9Onozawa Y. Iwahashi K. Yoshimoto K. et al.[Electroencephalographic background activity and blood biochemistry data in patients with chronic renal failure during chronic hemodialysis].Rinsho Byori. 2010; 58: 1169-1175PubMed Google Scholar These studies raise the possibility that the circadian timing system may be disrupted in CKD patients. The circadian system controls daily fluctuation of physiological functions in mammals. The central clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, regulates the peripheral clocks located in each organ to generate physiological rhythms.10Bass J. Takahashi J.S. Circadian integration of metabolism and energetics.Science. 2010; 330: 1349-1354Crossref PubMed Scopus (1394) Google Scholar, 11Albrecht U. Timing to perfection: the biology of central and peripheral circadian clocks.Neuron. 2012; 74: 246-260Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 12Tahara Y. Shibata S. Circadian rhythms of liver physiology and disease: experimental and clinical evidence.Nat Rev Gastroenterol Hepatol. 2016; 13: 217-226Crossref PubMed Scopus (168) Google Scholar In the cellular circadian clocks, CLOCK/BMAL1 works as a transcriptional activator to initiate transcription of the Per1/2/3 and Cry1/2 genes, and the PER/CRY complex inhibits transcriptional activity of CLOCK/BMAL1 on a cycle approximating 24 hours. Retinoic acid–related orphan receptor (ROR) and REV-ERBα/β activate and suppress Bmal1 transcription, respectively, to augment the 24-hour rhythm. Clock-controlled PAR-domain basic leucine zipper transcription factors albumin D-box binding protein (DBP), thyrotroph embryonic factor (TEF), and hepatic leukemia factor (HLF) are highly expressed in the kidney with circadian rhythmicity, and they regulate renal functional genes, such as key regulators of water and sodium balance including the vasopressin V2 receptor (V2r), aquaporin-2 (Aqp2), Aqp4, and endothelial sodium channel α (αENaC).13Zuber A.M. Centeno G. Pradervand S. et al.Molecular clock is involved in predictive circadian adjustment of renal function.Proc Natl Acad Sci U S A. 2009; 106: 16523-16528Crossref PubMed Scopus (219) Google Scholar It is well known that renal functions, including homeostatic control of water, electrolyte balance, and erythropoietin levels, show circadian rhythmicity.14Firsov D. Bonny O. Circadian regulation of renal function.Kidney Int. 2010; 78: 640-645Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Mice with a genetically disrupted circadian timing system (e.g., Clock mutant mice) exhibit both nocturia and nocturnal polyuria with lower expressions of several renal genes.15Ihara T. Mitsui T. Nakamura Y. et al.The Clock mutant mouse is a novel experimental model for nocturia and nocturnal polyuria.Neurourol Urodyn. 2017; 36: 1034-1038Crossref PubMed Scopus (22) Google Scholar These studies suggest that the circadian system is deeply involved in renal function. Adenine-induced tubulointerstitial nephropathy in rodents has been established as a strong model of renal dysfunction without the complications of surgery or increased mortality seen in other CKD models, such as unilateral ureteral obstruction and nephrectomy.16Jia T. Olauson H. Lindberg K. et al.A novel model of adenine-induced tubulointerstitial nephropathy in mice.BMC Nephrol. 2013; 14: 116Crossref PubMed Scopus (143) Google Scholar Normally, adenine is converted into the uric acid allantoin, which is excreted with urine. However, excess adenine accumulates and is converted into 8-dihydroxy adenine, then eventually 2,8-dihydroxy adenine, and these non-soluble materials crystallize in renal tubules and cause damage. After feeding of an adenine (0.2%)–containing casein-based diet for 2–4 weeks, inflammation and fibrosis are detected in the kidney, and the serum markers of renal dysfunction are elevated (BUN and creatinine).16Jia T. Olauson H. Lindberg K. et al.A novel model of adenine-induced tubulointerstitial nephropathy in mice.BMC Nephrol. 2013; 14: 116Crossref PubMed Scopus (143) Google Scholar However, the impact of adenine on circadian function is not known and is the focus of the present study. First, we analyzed the impact of CKD on locomotor activity, and video-analyzed sleep behavior, food/water intake, and urine volume. Next, we determined the impact of CKD on the rhythms of clock gene expression measured by PER2 bioluminescence. We also measured rhythms in gene expression using real-time polymerase chain reaction by sampling the kidney every 4 hours through the 24-hour cycle. Finally, we determined if the genetic disruption of the circadian clock (Clock mutant) rendered the mice more sensitive to renal damage due to the adenine diet. After 2 weeks of the adenine diet (Figure 1a), ICR mice showed decreased body weight compared with control mice (Figure 1b). Indicators of renal function, including BUN, serum and urine creatinine, and the anti-inflammatory hormone corticosterone, were found to be increased in the CKD group (Figure 1c–g). The amount and frequency of water intake was higher in both the day and night (Figure 1h). In contrast, food intake was not changed by adenine treatment (Figure 1i). As with drinking behavior, diminished day–night variations of urine total volume, sodium, and potassium were seen in the adenine-treated mice (Figure 1j). The disruption of the day–night difference in urine volume and its contents suggest that the circadian regulation of renal function is disrupted in the treated mice. Next, we measured daily rhythms of locomotor activity and sleep behavior as circadian output using infrared sensors (activity) and video-monitored immobility (sleep), respectively, using C57BL/6N male mice. At first, we confirmed serum and urine creatinine changes in this strain (Supplementary Figure S1A). Compared to control mice, the CKD mice showed less locomotor activity in the night, with an increased number of activity bouts (Figure 2a–c). A 2-way repeated measures analysis of variance run on the waveform (1 hour bins) confirmed significant effects of time (F[23, 322] = 63.56, P < 0.001) and treatment (F[1, 14] = 5.11, P < 0.001), and an interaction (F[23, 322] = 3.32, P < 0.001). The % activity in the light phase was also greater in the CKD group (11.5% ± 1.3% in the control group and 20.8% ± 2.1% in the CKD group, P < 0.01 by Student's t test). Despite these changes, the strength of the activity rhythm as measured by periodogram analysis did not vary with treatment (39.0% ± 1.6% in the control group and 32.8% ± 4.0% in the CKD group). A similar reduction of activity level and increase in activity bouts were seen in the male ICR strain (Supplementary Figure S2A–C). The amount of sleep behavior was not impacted by the treatment (Figure 2d and e). The CKD mice had a higher number of sleep bouts in their rest-phase (Figure 2f). Thus, the CKD mice exhibited reduced locomotor activity as well as more fragmented rhythms in sleep. Rhythms in activity and sleep are controlled by the SCN.12Tahara Y. Shibata S. Circadian rhythms of liver physiology and disease: experimental and clinical evidence.Nat Rev Gastroenterol Hepatol. 2016; 13: 217-226Crossref PubMed Scopus (168) Google Scholar Therefore, we next sought to determine whether the PER2 protein rhythms measured in the SCN were disrupted in the CKD mice. Brain slices containing the SCN were prepared from PER2::LUCIFERASE mice (ICR background) treated with the adenine diet. We found that the SCN from the CKD mice exhibited low-amplitude rhythms that damped faster than that in controls (Figure 3a–e). The endogenous circadian cycle length (period) was not altered, nor did we observe any differences in the peak phase of the bioluminescence rhythms (Figure 3d and e). Our data suggest that CKD weakens the amplitude but not the period or phase of the central circadian clock. Next, using in vivo imaging techniques,17Tahara Y. Kuroda H. Saito K. et al.In vivo monitoring of peripheral circadian clocks in the mouse.Curr Biol. 2012; 22: 1029-1034Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar we examined PER2::LUC bioluminescence from kidneys, liver, and submandibular gland throughout the 24-hour cycle (Figure 4a and b). We found that the daily average of bioluminescence and amplitude of the rhythms were reduced in all 3 tissues, whereas peak phase was unaltered (Figure 4c–e). We also measured PER2::LUC bioluminescence from kidney explants in vitro. Interestingly, we found that the rhythms in bioluminescence were not compromised in the CKD mice under these condition (Supplementary Figure S3). These findings suggest that the molecular clockwork in the intact organism is more impacted by CKD than the clockwork in the isolated kidney. Sex differences of CKD patients and CKD model animals are reported, with greater adverse effects in males.18Diwan V. Small D. Kauter K. et al.Gender differences in adenine-induced chronic kidney disease and cardiovascular complications in rats.Am J Physiol Renal Physiol. 2014; 307: F1169-F1178Crossref PubMed Scopus (55) Google Scholar,19Diwan V. Brown L. Gobe G.C. Adenine-induced chronic kidney disease in rats.Nephrology (Carlton). 2018; 23: 5-11Crossref PubMed Scopus (148) Google Scholar Consistently, compared with males (Figures 1 and 4), in female mice (Supplementary Figure S4), we found smaller effects on urine creatinine levels (71% reduction by adenine in females, 81% reduction in males) and in vivo renal PER2::LUC rhythms (31% reduction in females, 55% reduction in males). To get a more complete view, we used real-time polymerase chain reaction to measure the expression levels of clock genes sampled every 4 hours throughout the 24-hour cycle. The expression levels were reduced at several time points in all clock genes except for Dec1 and Cry1 (Figure 5a). In addition, mRNA levels of renal function genes (Scnn1a, Gilz, Aqp2, V2r), which were reported to be regulated by the circadian clock, also exhibited decreased expression (Figure 5b). Most of the circadian clock or clock-regulated genes that we examined showed significant rhythmicity in both control and CKD conditions (Supplementary Table S1). Some of them showed phase changes by CKD, but the direction of the phase changes depended on the genes (Supplementary Table S1). In contrast, inflammation (Mmp2, Mmp9, Il-1b) and fibrosis (Tgf-b1) markers were increased significantly throughout the day and night (Figure 5c). Similar results were seen in the C57BL/6N CKD mice for Rev-erba, Tef, and Tgf-b1, but not Per2 (Supplementary Figure S1). The gene expression data indicate that the molecular clock and its rhythmic outputs in the kidney are damped while inflammation is chronically increased in the CKD condition. We examined whether the renal gene expression changes in CKD mice would reverse themselves after a return to the normal diet from the adenine diet (Supplementary Figure S5). After 2 weeks of recovery, BUN and urine creatinine levels were slightly recovered but still significantly different from those in control mice. The expression level of clock genes (Per2, Clock, Rev-erba, Dbp) and the clock-controlled gene (Scnn1a) stayed dampened compared with the controls. Tgf-b1, a marker of fibrosis, showed constantly high levels of mRNA as well. These observations suggest that dampened molecular clocks persist after establishment of CKD. A variety of studies suggest that a robust circadian rhythm is important for health and that environmental or genetic disruptions of the circadian system can accelerate disease progression.12Tahara Y. Shibata S. Circadian rhythms of liver physiology and disease: experimental and clinical evidence.Nat Rev Gastroenterol Hepatol. 2016; 13: 217-226Crossref PubMed Scopus (168) Google Scholar,20Turek F.W. Joshu C. Kohsaka A. et al.Obesity and metabolic syndrome in circadian Clock mutant mice.Science. 2005; 308: 1043-1045Crossref PubMed Scopus (2029) Google Scholar,21Kettner N.M. Mayo S.A. Hua J. et al.Circadian dysfunction induces leptin resistance in mice.Cell Metab. 2015; 22: 448-459Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar Therefore, we examined the impact of Clock⊿19/⊿19 mutation, in which exon 19 (51 amino acids) is missing and dominant-negative protein is expressed,22Vitaterna M.H. King D.P. Chang A.M. et al.Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior.Science. 1994; 264: 719-725Crossref PubMed Scopus (1367) Google Scholar on the development of adenine-induced CKD (Figure 6, Figure 7, Figure 8). Clock mutant mice showed a significantly lower level of urine creatinine on days 9 and 13, and an earlier increase of serum BUN levels on days 5 and 9 compared with wild-type (WT) mice (Figure 6a and b). Serum creatinine or urine osmolarity were not different between genotypes (Figure 6c and d). Renal hematoxylin and eosin, and Masson trichrome, staining confirmed adenine-induced tubular damage and showed significantly higher 2,8-dehydroxyadenine deposition in Clock mutant mice compared to WT mice, but other factors (distal tubular dilation and fibrosis) were not different between genotypes (Figure 6e–g). As reported previously,13Zuber A.M. Centeno G. Pradervand S. et al.Molecular clock is involved in predictive circadian adjustment of renal function.Proc Natl Acad Sci U S A. 2009; 106: 16523-16528Crossref PubMed Scopus (219) Google Scholar,15Ihara T. Mitsui T. Nakamura Y. et al.The Clock mutant mouse is a novel experimental model for nocturia and nocturnal polyuria.Neurourol Urodyn. 2017; 36: 1034-1038Crossref PubMed Scopus (22) Google Scholar Clock mutant mice showed a higher volume of total urine (corrected by body weight) with increased daytime urine excretion in the control diet condition (Figure 6h). The treated mutants exhibited significantly increased urine excretion and urine sodium/potassium content during both day and night, compared with WT CKD mice (Figure 6h). After 2 weeks of adenine treatment, daily gene expression changes were measured (Figure 7a and Supplementary Figure S6A). Mmp2/9, but not Tgf-b1, expression was significantly increased compared with that in WT mice in CKD conditions. For Mmp2, a 2-way analysis of variance revealed significant effects of treatment (F[1, 60] = 167.2, P < 0.001) and genotype (F[1, 60] = 10.45, P < 0.01), and an interaction (F[1, 60] = 4.18, P < 0.05). For Mmp9, a 2-way analysis of variance confirmed significant effects of treatment (F[1, 60] = 106.5, P < 0.001) and genotype (F[1, 60] = 29.47, P < 0.001), and an interaction (F[1, 60] = 4.81, P < 0.05). Although the 2,8-dehydroxyadenine deposition was higher in the mutant, Xanthine dehydrogenase (Xdh), which produces insoluble 2,8-dehydroxyadenine, was increased in the liver in both adenine-treated WT and mutant mice with no genotype difference (Figure 7a). Consistently, matrix metalloproteinase 2 (MMP2) protein level and gelatinase activity of pro-MMP2 were higher in the mutant mice, measured by western blotting and zymography, respectively (Figure 7b–d). In summary, our data suggest that the Clock mutation potentiated the progression of adenine-induced CKD and led to worsening of symptoms due to the higher 2,8-dehydroxyadenine deposition and MMP2 expression.Figure 7Matrix metalloproteinase (MMP)–2 and –9 expressions of adenine-induced chronic kidney disease (CKD) in Clock (C) mutant mice. (a) mRNA gene expressions (Mmp-2, Mmp-9, and Tgf-β1 in the kidney; Xdh in the liver) after 2 weeks of adenine treatment in wild-type (WT; W) and Clock mutant mice. Samples were taken at ZT6, Z12, Z18, and Z24 (Zeitgeber time; ZT0 is the time of light on and ZT12 is the time of light off; n = 4 in each time point) but the all–time point samples were combined. Daily change of these gene expressions are shown in Supplementary Figure S6. (b,c) Western blotting analysis of MMP-2 (b) and MMP-9 (c) in the kidney are shown with representative band images (left) and relative values (right, 1 as WT mice with control [Ctl] diet). (d) Gelatin zymography of MMP-2 and MMP-9 in the kidney with representative band images (left) and relative values (right, 1 as WT mice with Ctl diet). n = 3 in Ctl, n = 6 in CKD for western blotting and zymography. All values are expressed as individual plots with mean ± SEM. P values, versus WT: *P < 0.05; **P < 0.01; ***P < 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Blood pressure (BP) measurement of adenine-induced chronic kidney disease (CKD) in Clock (C) mutant mice. (a) Averaged systolic (Sys), mean, and diastolic (Dia) BP, heart rate (beats per minute [bpm]), and movement (activity) measured by the radiotelemetry method after 2 weeks of control (Ctl) or adenine (CKD) diet treatment are shown. n = 4 for Ctl, n = 3 for CKD in each genotype. Percent difference of each parameter between day and night is also shown. Individual plots are shown in Supplementary Figure S6B. Results of cosinor analysis and 2-way repeated measures analysis of variance are shown in Supplementary Tables S2 and S3, respectively. (b) Averaged Sys, mean, and Dia BP, and heart rate measured by tail-cuff method at ZT14–18 (Zeitgeber time; ZT0 is the time of light on and ZT12 is the time of light off; data measured at ZT4–8 are shown in Supplementary Figure S6C). All values are expressed as individual plots with mean ± SEM. P values, versus wild-type (WT; W): *P < 0.05; **P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effects of adenine treatment and Clock mutation on heart rate and blood pressure were examined using a telemetry system and the tail-cuff method (Figure 8). Under control conditions, the Clock mutant mice showed a phase delayed rise in diurnal rhythm in blood pressure, heart rate, and activity (Figure 8a; cosinor analysis and 2-way repeated measures analysis of variance are indicated in Supplementary Tables S2 and S3, respectively), as previously reported.23Sei H. Oishi K. Chikahisa S. et al.Diurnal amplitudes of arterial pressure and heart rate are dampened in Clock mutant mice and adrenalectomized mice.Endocrinology. 2008; 149: 3576-3580Crossref PubMed Scopus (50) Google Scholar After 2 weeks of adenine treatment, systolic blood pressure was increased compared to controls, and the normal day–night difference was disrupted with non-dipping at rest phase, analyzed by the goodness-of-fit value of cosinor analysis and the percent difference between day and night (Figure 8a; Supplementary Tables S2 and S3). Hypertension was confirmed using a tail-cuff blood pressure measurement, and the mutant CKD mice showed higher blood pressure compared to WT CKD mice at ZT14-18 (Figure 8b). The heart rate in the mutant mice was lower than that in controls and exhibited a disrupted daily rhythm (Figure 8a). Similar to the locomotor activity data measured by infrared sensor (Figure 2a–c and Supplementary Figure S2), activity levels as measured by telemetry sensor were reduced in CKD condition (Figure 8a). Therefore, Clock mutant mice showed higher blood pressure and lower heart rate than WT mice under adenine treatment, confirming that Clock mutation makes mice vulnerable to the impact of the adenine diet. This study used the adenine-induced model of kidney damage to determine whether CKD can impact the circadian timing system. We observed that the treated mice exhibited fragmented rhythms in activity and sleep, and reduction of overall activity level. We found significant reductions in the amplitude of the PER2-driven bioluminescence rhythms in the SCN in vitro and in the kidney in vivo. Further analysis of gene expression in the kidney using real-time polymerase chain reaction confirmed that amplitudes of the oscillations in a number of circadian-regulated transcripts were reduced. The CKD model also exhibited clear evidence of renal inflammation and distal tubular dilation. Non-dipping hypertension with disrupted circadian rhythm and low heart rate was seen in the CKD model mice, confirming the circadian disruption in this model and similarity with human CKD patients.24Biyik Z. Yavuz Y.C. Altintepe L. et al.Nondipping heart rate and associated factors in patients with chronic kidney disease.Clin Exp Nephrol. 2019; 23: 1298-1305Crossref PubMed Scopus (7) Google Scholar Finally, Clock mutant mice with a dysfunctional circadian timing system proved to be more sensitive to adenine-induced kidney damage than were WT controls, which is caused by higher adenine metabolite deposition and higher MMP expressions in the kidney. Previous papers have shown that renal disease patients have sleep problems, including fragmentation of sleep6Agarwal R. Light R.P. Sleep and activity in chronic kidney disease: a longitudinal study.Clin J Am Soc Nephrol. 2011; 6: 1258-1265Crossref PubMed Scopus (58) Google Scholar and daytime sleepiness7Hanly P. Sleep apnea and daytime sleepiness in end-stage renal disease.Semin Dial. 2004; 17: 109-114Crossref PubMed Scopus (101) Google Scholar with reduced duration and intensity of daytime physical activity.6Agarwal R. Light R.P. Sleep and activity in chronic kidney disease: a longitudinal study.Clin J Am Soc Nephrol. 2011; 6: 1258-1265Crossref PubMed Scopus (58) Google Scholar We found that the adenine-treated mice did not exhibit extensive disruption or increase of their rhythm in sleep. We did see clear evidence of fragmentation of the rhythms in sleep and activity with decreased cage activity (Figure 2; Supplementary Figure S2). Prior work in another CKD model (5/6 nephrectomy rats) found decreased cage activity and increased rapid eye movement/non–rapid eye movement (REM/NREM) sleep in the end of the active period, but it did not examine the fragmentation of sleep and activity.25Hsu C.Y. Chang F.C. Ng H.Y. et al.Disrupted circadian rhythm in rats with nephrectomy-induced chronic kidney disease.Life Sci. 2012; 91: 127-131Crossref PubMed Scopus (14) Google Scholar Fragmented sleep in the rest period could be due to the arousal signals of urine excretion or thirst in the CKD mice, as the treated