**4. Chronodisruption in CKD**

Several alterations of circadian rhythms are well characterized in CKD patients and there is accumulating evidence that at least some of them may adversely affect health, thus fulfilling criteria to be considered chronodisruption. These include disordered sleep, non-dipping hypertension, failure to properly concentrate urine at night and the circadian pattern of proteinuria in patients with nephrotic syndrome: Peak protein excretion occurs at around 16.00 h and the nadir at 03.00 h and is independent of GFR [14]. Ultradian rhythms have also been described in CKD. For example, in patients with end-stage renal disease treated with hemodialysis, blood pressure varies seasonally, with higher values in the winter and lower values in the summer [30]. However, this pattern has not been directly compared to that of non-CKD individuals. Of the diverse altered circadian rhythms in CKD, the ones best characterized with adverse consequences for health, meeting the criterion to define chronodisruption are disordered sleep and non-dipping hypertension and will be discussed more extensively.

Sleep timing, quality and/or duration are frequently disturbed in CKD patients and this can be reproduced by subtotal nephrectomy in rats [26] or in mice with adenine-induced CKD [21]. In patients with mild to moderate CKD, lower eGFR was associated with shorter sleep duration (−1.1 mL/min/1.73 m<sup>2</sup> per hour less sleep), greater sleep fragmentation (−2.6 mL/min/1.73 m<sup>2</sup> per 10% higher fragmentation) and later timing of sleep (−0.9 mL min/1.73 m<sup>2</sup> per hour later). Higher proteinuria was also associated with greater sleep fragmentation (approximately 28% higher per 10% higher fragmentation) [31]. However, from these studies, potential causality and direction of the association is unclear, since CKD may cause chronodisruption but chronodisruption may theoretically lead to CKD progression. The nocturnal melatonin peak appears to be preserved just in nocturnal hemodialysis patients but not in patients on other dialysis modalities. In this regard, exogenous melatonin may improve intrarenal renin angiotensin system activation and renal injury in experimental CKD [32]. Specific conditions associated to CKD may contribute to disrupted sleep patterns. These include nocturia elated to decreased urine concentration capacity and obstructive sleep apnea. The prevalence of obstructive sleep apnea increases as kidney function declines and is higher among patients with ESRD. obstructive sleep apnea may contribute to higher nocturnal blood pressure and to pulmonary hypertension and these may improve on continuous positive airway pressure (CPAP) [33–35].

In CKD patients, the prevalence of reverse dipping (night-time blood pressure peak) for systolic blood pressure and episodes of hypotension during daytime is doubled, independently of blood pressure control [36]. Uninephrectomy by itself interfered with blood pressure rhythms. Albuminuria in hypertensive patients is also accompanied by quantitatively striking higher nighttime systolic blood pressure, particularly in patients with diabetes with very high albuminuria and low eGFR [37]. Although studies regarding causality are needed, this observation may point out to a CKD A2/A3-dependent altered clock: That is, albuminuria itself may potentially be a chronodisruptor, even when global kidney function (GFR) is preserved, on top of any potential chronodisruptor activity of uremic toxins that accumulate when GFR falls. Further supporting a potential role of albuminuria itself, in minimal change nephrotic syndrome patients with overall preserved GFR (around 75 mL/min/1.73 m2), sleeping/waking systolic and diastolic blood pressure ratios were higher than in healthy controls and this was reversed by remission of proteinuria [38].

Non-dipping is a recognized cardiovascular risk factor. In the general population, there is a linear relationship between the nocturnal decline in blood pressure and cardiovascular mortality. On average, each 5% decrease in the decline in nocturnal systolic/diastolic blood pressure was associated with an approximately 20% greater risk of cardiovascular mortality and this was observed even when 24-h blood pressure values were within the normal range (average 118/69 mmHg), diminished nocturnal decreases [39]. In CKD patients this may be magnified, as they have higher systolic blood pressure during the night-time and greater prevalence of non-dipping. Indeed, nocturnal systolic blood pressure correlated more strongly with cardiac organ damage [40]. In hemodialysis patients, increased short-term nighttime pulse pressure variability but not ambulatory blood pressure levels were significantly predictive of long-term all-cause mortality [41].

Several individual contributors to the circadian regulation of blood pressure have been identified and these include local kidney molecular clocks, whose local expression may be potentially altered by kidney disease mediators. Thus, *Bmal1* deficiency in juxtaglomerular renin-secreting granular cells resulted in polyuria, changes in the circadian rhythm of urinary sodium excretion, increased GFR, and lower plasma aldosterone levels and lower blood pressure [42]. The sodium-chloride cotransporter (NCC, SLC12A3) in distal convoluted tubules contributes to sodium balance and blood pressure regulation. Disturbing this rhythm induces "nondipping" blood pressure. Both mineralocorticoids and glucocorticoids regulate NCC activity. Mineralocorticoid receptor activation maintains the NCC protein pool while glucocorticoid receptor activation regulates NCC phosphorylation and the diurnal rhythm of NCC activity [43]. ATP1B1 encodes the β1 subunit of the Na+/K<sup>+</sup>-ATPase. Atp1b1 mRNA and protein levels in mouse kidney have a circadian rhythm that was antiphasic to the blood pressure rhythm. In *Dec1*-deficient mice, kidney Atp1b1 expression was increased and blood pressure was

lower. In contrast, in *Clock*-mutant mice, Atp1b1 expression was low and blood pressure high [44]. The expression of both NCC and ATP1B1 is altered in kidney injury, potentially linking kidney injury to an altered expression of kidney circadian genes regulating blood pressure [45,46].

The location of disrupted timekeeping in CKD merits further study. In murine adenine-induced CKD, in vivo disrupted timekeeping could be dissociated in vitro into a suprachiasmatic nucleus pacing, which remained uncompromised, and a kidney clock that became a less robust circadian oscillator with a longer period, suggesting that the kidney contributes to overall circadian timekeeping and that there is local kidney disruption of circadian rhythms during CKD [47]. By contrast, in vivo exploration of mice with adenine-induced CKD disclosed low amplitude PER2:luciferase rhythms in their central suprachiasmatic nucleus circadian clock and in intact kidney, liver, and submandibular gland, as well as altered expression patterns of circadian genes including canonical clock genes and kidney genes such as Hif, Aqp2, and V2r [21]. Overall, these results point to interference of peripheral clocks with the central clock in CKD.

Failure to properly concentrate urine at night may further aggravate CKD-associated sleep disruption through nocturia. However, there are potentially more severe consequences. Thus, improper water excretion will promote the secretion of antidiuretic hormone (vasopressin, ADH). There is increasing evidence that overactivation of ADH may be detrimental. Specifically, the vasopressin 2 receptor (V2R) blocker tolvaptan slows the progression of autosomal dominant polycystic kidney disease (ADPKD) [48]. While this was initially thought to be related to kidney cys<sup>t</sup> specific intracellular signaling events, an adverse impact of ADH on glomerular hyperfiltration was later identified that may be a universal driver of CKD progression, not limited to ADPKD [49,50]. In this regard, circulating copeptin levels provide a better understanding of ADH activation that measuring ADH itself, which is short lived. Serum copeptin is increased in hypertension, CKD and cardiovascular disease, and ADH activation of V1R and/or V2R may be detrimental to the kidney and the cardiovascular system [51].

The altered circadian pattern of proteinuria may impact the assessment of the severity or proteinuria when different timed urine samples are assessed (12 h vs. 24 h vs. point collections), but whether this leads to any health consequence is currently unclear.

CKD has a bidirectional relationship with aging. On one hand, aging is associated with a progressive decrease in GFR. On the other, CKD causes accelerated aging and some of the factors responsible for this phenotype, such as decreased production of the anti-aging factor Klotho have been identified, as discussed below. Interestingly, aging is associated with altered central and peripheral circadian rhythms, and the sleep–wake cycle [52], leading to a phase advance, rhythm fragmentation and flattening [53]. This may in part be offset by regular physical activity [52]. Given the close association of CKD with aging, further studies are required that explore to what extent the age-associated loss of renal function contributes to age-associated circadian rhythm abnormalities and age-associated organ dysfunction and disease.

### **5. Chronodisruptors as Therapeutic Targets in CKD**

A PubMed search for "chronodisruptors" in January 2020 resulted in only 5 hits, none of them related to CKD. This may relate to both limited understanding of chronodisruptors as with limited use of the term.

Identifying and targeting chronodisruptors may identify novel approaches to the prevention and therapy of CKD. Potential chronodisruptors include diet, the light–dark cycle, inflammatory mediators, uremic toxins, HIF abnormalities, and physical inactivity. We will briefly discuss examples of all of these (summarized in Table 2). While diet, light clues and inflammation may be active at all stages of CKD, even before GFR decreases, accumulation or uremic toxins would be expected to be active only after significant decrease of GFR has taken place, i.e., after significant loss of kidney mass.


**Table 2.** Examples of potential chronodisruptors in chronic kidney disease (CKD) patients.
