*5.4. Uremic Toxins*

A key feature of advanced CKD is the accumulation of uremic retention solutes, molecules usually excreted by the kidneys that accumulate in the circulation when GFR decreases [11]. Some of these uremic retention solutes have a clear adverse impact on pathophysiological processes, promoting CKD progression and manifestations, they are the so-called uremic toxins. When kidneys fail, renal function is replaced by dialysis or eventually by a kidney graft. Unfortunately, while dialysis prevents acute uremic death, it provides only a very limited capacity to clear uremic toxins, especially those of gu<sup>t</sup> origin that circulate bound to serum proteins, which may be of special interest from the point of view of chronodisruption. Thus, several gut-derived uremic toxins bind and activate the Aryl Hydrocarbon Receptor (AhR). These include uremic toxins derived from tryptophan, some of gu<sup>t</sup> microbiota origin, such as indolic uremic toxins (indoxyl sulfate, indole-3 acetic acid, and indoxyl-β-d-glucuronide) and uremic toxins from the kynurenine pathway (kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and quinolinic acid) [83,84]. Interestingly, AhR exhibits a rhythmic expression and time-dependent sensitivity to activation by AhR agonists and in response to at least some ligands, AhR forms a heterodimer with *Bmal1* and inhibits *Clock*/*Bmal1* activity, modulating amplitude and phase of rhythms in circadian clock genes [85,86]. In this regard, AhR deficiency enhanced behavioral responses to changes in the light–dark cycle, increased rhythmic amplitude of circadian clock genes in the liver, and altered glucose and insulin rhythms [86].

Kidney proximal tubule cells sense elevated endogenous, gu<sup>t</sup> microbiome-derived, uremic retention solutes which elicit a compensatory response consisting of up-regulating the organic anion transporter-1 (OAT1), thus increasing metabolite secretion in urine [87]. This was clearly illustrated for indoxyl sulfate which induced OAT1 expression via AhR and EGFR signaling, controlled by miR-223 [87]. AhR protein expression was additionally positively associated with plasma levels of another indolic uremic toxin, indole-3 acetic acid (IAA) [88]. IAA is responsible for some adverse effects potentially related to the increased cardiovascular risk of CKD patients, such as increasing the expression of tissue factor in human vascular cells via the AhR [89]. However, up to now it is unknown to what extent the circadian expression of AhR is disrupted in CKD, what role might uremic toxins and the microbiota have in this phenomenon and what the consequences in any alterations in this system circadian regulation might be for CKD patients.

### *5.5. Disrupted HIF Activation and EPO Production*

Hypoxia-inducible factor (HIF) are a family of transcription factors that protect from hypoxia both at the local, autocrine/paracrine level and by driving erythropoietin production, also through an endocrine mediator of kidney origin. Thus, the kidney has the lowest pO2 in the body, a consequence of the existence of two consecutive capillary networks (glomerular and peritubular) and of the high metabolic rate of tubular cells which spend huge amounts of energy in recovering filtered molecules. This is the likely reason for the kidney location of erythropoietin-producing cells, a key defense mechanism against hypoxia that modulates hemoglobin availability and, thus, oxygen transport capacity by red blood cells.

The expression of a key HIF protein, HIF1<sup>α</sup>, is under circadian rhythm control. CRY1 reduces HIF-1α half-life and HIF binding to target gene promoters and abrogation of CRY1/2 stabilized HIF1α in response to hypoxia [90] while PER2 activates HIF-1α and facilitates its recruitment to promoter regions of its downstream genes. HIF-1α activation by PER2 was related to keeping the asparagine residue at position 803 of HIF-1α (HIF-1α N803) unhydroxylated by hypoxic stimulation in the absence of changes in HIF-1α protein levels [91]. In murine heart ischemia, *Per2* was required for Hif-1α stabilization [92]. This may be exploited therapeutically. Thus, *Per2* stabilization through adenosine activation of Adora2b or by exposure to intense light modified HIF-dependent cardiac metabolism, resulting in the transcriptional induction of glycolytic enzymes and *Per2*-dependent protection from ischemia [92]. So far, no such experiments have been reported for kidney disease. By contrast, BMAL1 deficiency increased HIF1α protein levels under hypoxic conditions. Induction of clock and HIF1α target genes in response to strenuous exercise varied according to the time of day in wild-type mice. Thus, interactions between circadian and HIF pathways influence metabolic adaptation to hypoxia [93].

Circadian transgenic zebrafish cells simulating a repressed or an overstimulated circadian clock, resulted in altered gene transcription levels of oxygen-regulated genes such as *EPO* and altered the hypoxia-induced increase in Hif-1α protein concentration. The amount of Hif-1α protein accumulated during the hypoxic response depended on the time of the day, with one maximum during the light phase and a second one during the dark phase [94].

The positive effects of HIF prolyl hydroxylase inhibitors (that is, HIF activators) over anemia and other cardiovascular risk parameters in CKD patients [95] raises the possibility that downregulation of HIF activation righter than loss of renal mass is a key driver of uremic anemia and may allow the exploration of the chronodisruption impact of uremic anemia itself.
