A molecular circadian clock operates in the parathyroid gland and is disturbed in chronic kidney disease associated bone and mineral disorder

Circadian rhythms in metabolism, hormone secretion, cell cycle and locomotor activity are regulated by a molecular circadian clock with the master clock in the suprachiasmatic nucleus of the central nervous system. However, an internal clock is also expressed in several peripheral tissues. Although about 10% of all genes are regulated by clock machinery an internal molecular circadian clock in the parathyroid glands has not previously been investigated. Parathyroid hormone secretion exhibits a diurnal variation and parathyroid hormone gene promoter contains an E-box like element, a known target of circadian clock proteins. Therefore, we examined whether an internal molecular circadian clock is operating in parathyroid glands, whether it is entrained by feeding and how it responds to chronic kidney disease. As uremia is associated with extreme parathyroid growth and since disturbed circadian rhythm is related to abnormal growth, we examined the expression of parathyroid clock and clock-regulated cell cycle genes in parathyroid glands of normal and uremic rats. Circadian clock genes were found to be rhythmically expressed in normal parathyroid glands and this clock was minimally entrained by feeding. Diurnal regulation of parathyroid glands was next examined. Significant rhythmicity of fibroblast-growth-factor-receptor-1, MafB and Gata3 was found. In uremic rats, deregulation of circadian clock genes and the cell cycle regulators, Cyclin D1, c-Myc, Wee1 and p27, which are influenced by the circadian clock, was found in parathyroid glands as well as the aorta. Thus, a circadian clock operates in parathyroid glands and this clock and downstream cell cycle regulators are disturbed in uremia and may contribute to dysregulated parathyroid proliferation in secondary hyperparathyroidism. Circadian rhythms in metabolism, hormone secretion, cell cycle and locomotor activity are regulated by a molecular circadian clock with the master clock in the suprachiasmatic nucleus of the central nervous system. However, an internal clock is also expressed in several peripheral tissues. Although about 10% of all genes are regulated by clock machinery an internal molecular circadian clock in the parathyroid glands has not previously been investigated. Parathyroid hormone secretion exhibits a diurnal variation and parathyroid hormone gene promoter contains an E-box like element, a known target of circadian clock proteins. Therefore, we examined whether an internal molecular circadian clock is operating in parathyroid glands, whether it is entrained by feeding and how it responds to chronic kidney disease. As uremia is associated with extreme parathyroid growth and since disturbed circadian rhythm is related to abnormal growth, we examined the expression of parathyroid clock and clock-regulated cell cycle genes in parathyroid glands of normal and uremic rats. Circadian clock genes were found to be rhythmically expressed in normal parathyroid glands and this clock was minimally entrained by feeding. Diurnal regulation of parathyroid glands was next examined. Significant rhythmicity of fibroblast-growth-factor-receptor-1, MafB and Gata3 was found. In uremic rats, deregulation of circadian clock genes and the cell cycle regulators, Cyclin D1, c-Myc, Wee1 and p27, which are influenced by the circadian clock, was found in parathyroid glands as well as the aorta. Thus, a circadian clock operates in parathyroid glands and this clock and downstream cell cycle regulators are disturbed in uremia and may contribute to dysregulated parathyroid proliferation in secondary hyperparathyroidism. Translational StatementCircadian rhythms in metabolism, hormone secretion, cell cycle, and locomotor activity are regulated by a molecular circadian clock. Disruption of the circadian rhythm may cause serious health problems and abnormal cell growth. Secondary hyperparathyroidism is a severe complication of chronic kidney disease associated with increased morbidity and mortality, and the control of parathyroid hyperplasia is a clinical challenge. This article describes the existence of an internal molecular circadian clock that is operating in the parathyroid gland and which is disturbed in chronic kidney disease. Deregulation of the circadian clock in the parathyroids might therefore potentially call for the use of chronotherapy in future clinical studies. Circadian rhythms in metabolism, hormone secretion, cell cycle, and locomotor activity are regulated by a molecular circadian clock. Disruption of the circadian rhythm may cause serious health problems and abnormal cell growth. Secondary hyperparathyroidism is a severe complication of chronic kidney disease associated with increased morbidity and mortality, and the control of parathyroid hyperplasia is a clinical challenge. This article describes the existence of an internal molecular circadian clock that is operating in the parathyroid gland and which is disturbed in chronic kidney disease. Deregulation of the circadian clock in the parathyroids might therefore potentially call for the use of chronotherapy in future clinical studies. Circadian rhythms are controlled by the molecular clock mechanisms and have a systemic impact on all aspects of physiology, including hormonal secretion, metabolism, and cell cycle. Core clock genes and their products are involved in transcriptional-translational feedback loops and are revealed as important regulators of organ function. The possible existence of an internal circadian clock machinery in the parathyroid gland has not previously been examined. 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Usui T. et al.Transcription factor MafB may play an important role in secondary hyperparathyroidism.Kidney Int. 2018; 93: 54-68Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar,43Kebebew E. Peng M. Wong M.G. et al.GCMB gene, a master regulator of parathyroid gland development, expression, and regulation in hyperparathyroidism.Surgery. 2004; 136: 1261-1266Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar The present study is the first to investigate the internal molecular circadian clock in the parathyroid gland and whether such an internal molecular circadian clock is related to the circadian rhythm of p-PTH and parathyroid function. Furthermore, we examine the potential role of deregulation of such a circadian clock in the development of sHPT in CKD–mineral and bone disorder (MBD) and expand the impact of deregulation of the clock in CKD-MBD to uremic vasculopathy. Rats kept in 12:12 hour light-dark cycle and fed ad libitum were sacrificed at 4-hour intervals for 24 hours and parathyroid glands were investigated for gene and protein expression of core clock components (protocol 1). Rhythmicity of the gene expression was assessed by cosinor analysis, fitted to a periodicity of 24 hours and was significant for Bmal1 (P < 0.0001), Npas2 (P < 0.0001), Per1 (P < 0.02), Per2 (P < 0.0001), Per3 (P < 0.0001), Cry1-2 (P < 0.0001), and Rev-Erbα (P < 0.001). No significant circadian rhythm was found for CK1ε and Clock (Figure 1). JTK_CYKLE (Hughes Lab, Pulmonary and Critical Care Medicine, Washington University School of Medicine, St. Louis, MO) analysis confirmed the significant circadian rhythmicity of core clock genes (Table 1).Table 1Circadian parameters of core clock genes estimated by JTK_CYCLE analysis in the parathyroids from controls, CKD rats, and rats exposed to restricted feedingMesor (CI)Amplitude (CI)Acrophase (ZT) (CI)Period (h)P value (JTK)ControlBmal10.73 (0.58, 0.89)0.66 (0.44, 0.88)0.48 (23.27, 1.69)24<0.0001Clock0.79 (0.69, 0.89)0.05 (−0.09, 0.20)8.65 (22.62, 18.68)161Npas20.88 (0.69, 1.08)0.64 (0.36, 0.91)0.20 (22.61, 1.79)24<0.0001Per10.71 (0.58, 0.83)0.24 (0.06, 0.42)10.72 (8.01, 13.43)240.57Per20.89 (0.73, 1.05)0.72 (0.49, 0.94)14.82 (13.61, 16.03)24<0.0001Per30.82 (0.66, 0.98)0.64 (0.40, 0.87)11.64 (10.28, 12.99)24<0.0001Cry10.99 (0.86, 1.12)0.61 (0.43, 0.79)18.17 (16.98, 19.36)20<0.0001Cry20.90 (0.79, 1.00)0.42 (0.26, 0.57)14.07 (12.67, 15.47)240.0001Rev-Erbα0.47 (0.29, 0.65)0.62 (0.37, 0.88)8.61 (7.06, 10.16)24<0.0001CK1ε0.78 (0.64, 0.92)0.11 (−0.09, 0.31)15.52 (8.665, 22.38)121CKDBmal10.79 (0.70, 0.87)0.64 (0.52, 0.76)1.22 (0.55, 1.89)24<0.0001Clock0.90 (0.81, 1.00)0.14 (0.004, 0.28)3.18 (23.46, 6.90)120.27Npas20.57 (0.49, 0.66)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.0.52 (0.40, 0.64)2.89 (2.02, 3.76)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.24<0.0001Per11.08 (0.94, 1.22)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.0.19 (−0.02, 0.40)10.82 (6.84, 14.80)120.16Per21.41 (1.10, 1.72)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.0.96 (0.52, 1.40)15.72 (13.92, 17.52)20<0.0001Per31.11 (0.87, 1.34)1.20 (0.86, 1.54)14.13 (13.10, 15.16)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.20<0.0001Cry11.11 (0.92, 1.31)0.71 (0.43, 0.99)20.77 (19.44, 22.11)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.20<0.0001Cry21.10 (0.95, 1.25)0.40 (0.17, 0.62)13.38 (11.36, 15.40)200.0005Rev-Erbα1.27 (1.04, 1.50)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.1.76 (1.42, 2.09)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.9.5 (8.79, 10.21)24<0.0001CK1ε0.96 (0.83, 1.09)0.12 (−0.06, 0.29)6.93 (0.59, 13.27)160.017RFBmal10.68 (0.54, 0.81)0.35 (0.16, 0.54)23.82 (21.78, 25.86)16<0.0001Clock0.78 (0.67, 0.89)0.03 (−0.13, 0.18)23.89 (0.00, 23.99)161Npas20.63 (0.51, 0.76)0.15 (−0.03, 0.34)aThere is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis.20.94 (16.55, 1.33)200.010Per10.84 (0.73, 0.94)0.21 (0.06, 0.37)14.94 (12.17, 17.71)240.46Per21.04 (0.87, 1.21)0.56 (0.32, 0.80)13.67 (12.04, 15.30)240.00093Per31.21 (0.88, 1.55)0.83 (0.35, 1.31)9.74 (7.61, 11.87)240.00039Cry10.99 (0.80, 1.17)0.36 (0.11, 0.61)16.91 (14.10, 19.72)240.055Cry21.13 (0.85, 1.40)0.67 (0.27, 1.06)12.78 (10.58, 14.98)240.019Rev-Erbα0.68 (0.50, 0.87)0.47 (0.21, 0.73)6.13 (3.92, 8.34)240.00051CK1ε0.84 (0.69, 0.98)0.04 (−0.17, 0.24)4.54 (0.00, 23.99)121CI, confidence interval; CKD, chronic kidney disease; RF, restricted feeding; ZT, Zeitgeber time (measured in hours since lights on). Data shown as mean (95% CI).a There is a significant difference when compared with control (P < 0.05). P value (JTK) is the adjusted P value reported from JTK_CYKLE analysis. Open table in a new tab CI, confidence interval; CKD, chronic kidney disease; RF, restricted feeding; ZT, Zeitgeber time (measured in hours since lights on). Data shown as mean (95% CI). Western blot showed significant circadian rhythmicity of core clock protein BMAL1 in the parathyroid glands (P < 0.05) with a phase delay of approximately 4 hours compared with that of the gene expression profile of Bmal1 (Supplementary Figure S1). Further support for an operating circadian clock on the protein level was found in our phosphoproteomics study with CK1E and CLOCK identified in the rat parathyroid glands (data not shown). P-PTH exhibited diurnal variation as previously shown by us and others,10el-Hajj Fuleihan G. Klerman E.B. Brown E.N. et al.The parathyroid hormone circadian rhythm is truly endogenous—a general clinical research center study.J Clin Endocrinol Metab. 1997; 82: 281-286Crossref PubMed Scopus (128) Google Scholar,11Nordholm A. Egstrand S. Gravesen E. et al.Circadian rhythm of activin A and related parameters of mineral metabolism in normal and uremic rats.Pflugers Arch. 2019; 471: 1079-1094Crossref PubMed Scopus (8) Google Scholar and its statistical significance was confirmed by cosinor analysis (Figure 2a), revealing acrophase in the inactive period and nadir during the active period. Parathyroid expression of the Pth gene showed no diurnal variation (Figure 2d). The gene expression of important regulators of PTH secretion was examined: Casr, Vdr, Klotho (Kl), and Fgfr1. Fgfr1 expression showed significant rhythmicity (P < 0.05), whereas the expression of FGFR1 cofactor Klotho was not rhythmic. Likewise, Casr and Vdr had no significant rhythmicity (Figure 2). The gene expression of cell-cycle regulators under circadian clock influence was examined: Wee1, Cyclin D1, p27, and c-Myc (Figure 3). Wee1 expression showed significant rhythmicity (P < 0.0005), as did Cyclin D1 (P < 0.03), whereas no significant rhythmicity was found for p27 (Supplementary Figure S2) or c-Myc. For the regulators of parathyroid proliferation and PTH secretion, MafB, Gata3, and Gcm2, expression was examined (Figure 3), and significant rhythmicity was demonstrated for MafB (P < 0.003) and Gata3 (P < 0.03), but not for Gcm2. Parathyroid function was assessed by acute induction of hypocalcemia in vivo in the rat (protocol 3).3Lewin E. Wang W. Olgaard K. Reversibility of experimental secondary hyperparathyroidism.Kidney Int. 1997; 52: 1232-1241Abstract Full Text PDF PubMed Scopus (45) Google Scholar,12Lewin E. Almaden Y. Rodriguez M. et al.PTHrP enhances the secretory response of PTH to a hypocalcemic stimulus in rat parathyroid glands.Kidney Int. 2000; 58: 71-81Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,13Lewin E. Garfia B. Almaden Y. et al.Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats.Kidney Int. 2003; 64: 63-70Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Ethylene-bis(oxyethylene-nitrilo)tetraacetic acid infused into the femoral vein resulted in progressive hypocalcemia starting at either ZT4 (light phase) or ZT16 (dark phase). ZT refers to time since light onset (zeitgeber time; “time-giver”). Hypocalcemia was induced at similar rates in the 2 groups (Figure 4a). Samples were drawn from a catheter in femoral artery to determine the secretory response to hypocalcemia. PTH secretion did not differ significantly between the 2 time points (Figure 4b). Rats were acclimatized to the standard conditions of 12:12 hour light-dark cycle and ad libitum feeding for 2 weeks and blood samples were drawn, then feeding was restricted to the light phase (ZT2–ZT12) for 4 weeks, blood samples were repeated and parathyroid glands harvested every 4 hours for 24 hours and gene expression examined (protocol 2). Significant rhythmicity was confirmed for p-PTH with comparable acrophase timing to that found in protocol 1 when rats were fed ad libitum (P < 0.0002) (Figure 5a). Significant rhythmicity was found also for p-P (P < 0.0001) (Figure 5b) and p-urea (P < 0.0001) (data not shown). P-Ca (Figure 5d) and creatinine (data not shown) remained constant during the day. Feeding restricted to the inactive phase markedly shifted the phase of p-PTH and p-P (Figure 5a and b) and urea (not shown), now exhibiting significant circadian pattern that mirrors that of rats with continuous access to feeding (P < 0.0001). In restricted feeding, p-Ca gained significant rhythmici