Circadian rhythm of activin A and related parameters of mineral metabolism in normal and uremic rats

Abstract

Activin A is a new fascinating player in chronic kidney disease-mineral and bone disorder (CKD-MBD), which is implicated in progressive renal disease, vascular calcification, and osteodystrophy. Plasma activin A rises early in the progression of renal disease. Disruption of circadian rhythms is related to increased risk of several diseases and circadian rhythms are observed in mineral homeostasis, bone parameters, and plasma levels of phosphate and PTH. Therefore, we examined the circadian rhythm of activin A and CKD-MBD-related parameters (phosphate, PTH, FGF23, and klotho) in healthy controls and CKD rats (5/6 nephrectomy) on high-, standard- and low-dietary phosphate contents as well as during fasting conditions. Plasma activin A exhibited circadian rhythmicity in healthy control rats with fourfold higher values at acrophase compared with nadir. The rhythm was obliterated in CKD. Activin A was higher in CKD rats compared with controls when measured at daytime but not significantly when measured at evening/nighttime, stressing the importance of time-specific reference intervals when interpreting plasma values. Plasma phosphate, PTH, and FGF23 all showed circadian rhythms in control rats, which were abolished or disrupted in CKD. Plasma klotho did not show circadian rhythm. Thus, the present investigation shows, for the first time, circadian rhythm of plasma activin A. The rhythmicity is severely disturbed by CKD and is associated with disturbed rhythms of phosphate and phosphate-regulating hormones PTH and FGF23, indicating that disturbed circadian rhythmicity is an important feature of CKD-MBD.

Keywords: Activin A; CKD; Circadian rhythm; FGF23; Klotho; Phosphate.

Fig. 1

Plasma activin A exhibits circadian rhythmicity in healthy control rats, but the rhythm is obliterated by CKD. a Circadian rhythm of plasma activin A in healthy controls (red), PNX LP (gray), PNX SP (blue), and PNX HP rats (green). Controls showed circadian rhythm (p < 0.001) with a fourfold higher value at 20:00 compared with 14:00. The rhythm was obliterated in all PNX groups. b–e Cosinor analysis confirmed the existence of circadian rhythm in healthy controls, p < 0.01, with acrophase at 22:00 (b) and obliteration of rhythm in PNX LP (c), PNX SP (d), and PNX HP rats (e). f Non-fasting (black) and fasting (gray) plasma activin A levels in controls, PNX LP, PNX SP, and PNX HP rats. Fasting caused an increase in controls and PNX LP rats, but not in PNX SP or PNX HP rats. All PNX groups had higher non-fasting activin A levels as compared with controls. *p < 0.05 and **p < 0.01 (compared with non-fasting). ^##p < 0.01 and ^###p < 0.001 (compared with non-fasting controls). PNX 5/6 partial nephrectomy, LP low-phosphate diet, SP standard-phosphate diet, HP high-phosphate diet. Mean ± SEM (a, f). Cosinor fit (b–e).

Fig. 2

Plasma activin A correlated with plasma phosphate and FGF23 in CKD rats but correlations were absent in controls. a, b Correlation between activin A and phosphate in controls (a) and CKD rats (b) revealed a positive correlation in CKD but not in control rats. c, d Similarly, a correlation between activin A and FGF23 was absent in controls (c) but present in CKD rats (d). e, f No correlation could be demonstrated between activin A and PTH in either controls (e) or CKD rats (f)

Fig. 3

Plasma FGF23 exhibits circadian rhythm in healthy control rats, which is abolished or disturbed in CKD. a Circadian rhythm of plasma FGF23 in healthy controls (red), PNX LP (gray), PNX SP (blue), and PNX HP rats (green). Control rats showed significant circadian rhythm (p < 0.001). The rhythm was preserved (p < 0.0001) but severely disturbed in PNX HP rats with shift of acrophase. In both PNX LP and SP rats, the rhythm was abolished. b–e Circadian rhythm examined by cosinor analysis confirmed the findings of rhythmicity in healthy controls, p < 0.01, (b), and PNX HP rats, p < 0.05 (e) as well as obliteration in PNX LP (c) and PNX SP rats (d). Acrophase was shifted to 09:00 in the PNX HP group (e) compared with 13:00 in controls (b). f Non-fasting (black) and fasting (gray) plasma FGF23 levels in controls, PNX LP, PNX SP, and PNX HP rats. Fasting caused an increase in plasma FGF23 in all PNX groups but not in controls. *p < 0.05, **p < 0.01, and ***p < 0.001 (compared with non-fasting). ^##p < 0.01 and ^####p < 0.0001 (compared with non-fasting controls). ^&&&p < 0.001 and ^&&&&p < 0.0001. PNX 5/6 partial nephrectomy, LP low-phosphate diet, SP standard-phosphate diet, HP high-phosphate diet. Mean ± SEM (a, f). Cosinor fit (b–e).

Fig. 4

The circadian rhythm of plasma PTH in healthy control rats is obliterated or disturbed in CKD. a Circadian rhythm of plasma PTH in healthy controls (red), PNX LP (gray), PNX SP (blue), and PNX HP rats (green). Control rats exhibited circadian rhythm (p < 0.001). The rhythm was obliterated or disturbed in CKD rats with shift of acrophase in all PNX groups. b–e Circadian rhythm examined by cosinor analysis confirmed the rhythmicity in healthy controls, p < 0.0001, with acrophase at 12:00 (b), and revealed a significant circadian rhythm in both PNX LP, p < 0.05 (c) and PNX SP rats, p < 0.05 (d) but the rhythm was completely abolished in PNX HP rats. Both PNX LP and SP rats had disturbed rhythm with shift in acrophase to 06:00 (c) and 10:00 (d), respectively. f Non-fasting (black) and fasting (gray) plasma PTH levels in controls, PNX LP, PNX SP, and PNX HP rats. Fasting caused an increase in plasma PTH in controls and PNX SP rats. *p < 0.05 and **p < 0.01 (compared with non-fasting). ^##p < 0.01 and ^####p < 0.0001 (compared with non-fasting controls). ^&p < 0.05 and ^&&p < 0.01. PNX 5/6 partial nephrectomy, LP low-phosphate diet, SP standard-phosphate diet, HP high-phosphate diet. sHPT secondary hyperparathyroidism. Mean ± SEM (a, f). Cosinor fit (b–e)

Fig. 5

The circadian rhythm of plasma phosphate in healthy control rats is disturbed in CKD. a Circadian rhythm of plasma phosphate in healthy controls (red), PNX LP (gray), PNX SP (blue), and PNX HP rats (green). All groups showed significant circadian rhythm (control p < 0.0001, PNX LP p < 0.05, PNX SP p < 0.05, PNX HP p < 0.0001). The rhythm was clearly disturbed in CKD rats with peaks at 20:00 in all PNX group, compared with 14:00 in controls. b–e Circadian rhythm by cosinor analysis confirmed the rhythmicity in healthy controls, p < 0.001 (b), PNX LP, p < 0.05 (c), PNX SP, p = 0.05 (d), and PNX HP rats, p < 0.0001 (e). All PNX groups had a shift in acrophase from 16:00 in controls (b) to 19:00 in PNX LP (c), 17:00 in PNX SP (d), and 00:00 in PNX HP (e). f Non-fasting (black) and fasting (gray) plasma phosphate levels in controls, PNX LP, PNX SP, and PNX HP rats. Fasting caused an increase in plasma phosphate in all groups. *p < 0.05, **p < 0.01, and ***p < 0.001 (compared with non-fasting). ^##p < 0.01 and ^####p < 0.0001 (compared with non-fasting controls). ^&&&&p < 0.0001. PNX 5/6 partial nephrectomy, LP low-phosphate diet, SP standard-phosphate diet, HP high-phosphate diet. Mean ± SEM (a, f). Cosinor fit (b–e).

Fig. 6

Plasma klotho does not exhibit circadian rhythm in control or CKD rats. a Stable levels of plasma klotho were found in both healthy controls (red) and PNX HP rats (green). b Non-fasting (black) and fasting (gray) plasma klotho levels in controls and PNX HP rats. Fasting did not affect plasma klotho in either group. c, d Circadian rhythm by cosinor analysis confirmed the lack of rhythmicity in controls (c) and PNX HP rats (d). PNX 5/6 partial nephrectomy, HP high-phosphate diet. Mean ± SEM (a, b). Cosinor fit (b, d).