**10. Circadian regulation of EVs**

Circadian rhythms are seen in many physiological processes in several organ systems of various species and these rhythms are controlled by a master pacemaker located in the hypothalamus called the central suprachiasmatic nucleus and peripheral clocks in peripheral tissues. The circadian clocks within peripheral tissues not only regulate local physiological functions but they also contain essential core clock proteins such as Per1/2, Bmal1, Cry1/2, and Clock that work in concert to generate cell-autonomous oscillations and circadian rhythms.

Since the content of EVs represents a "snapshot" of a cell's internal environment at a given time, it is important to consider the regulation of EV biogenesis and cargo in a circadian dependent manner. Proteomic analysis of uEVs revealed these vesicles are enriched in at least 19 proteins that are associated with various renal diseases [53]. Proteins that were previously found to be packaged within uEVs include aquaporin-2, subunits of ENaC, the sodium chloride co-transporter (NCC) and the sodium potassium chloride co-transporter (NKCC2) [53–55]. Multiple studies have shown several of these proteins including ENaC and NCC are regulated by circadian clock proteins that are responsible for regulating circadian patterns and rhythm [56, 57].

At least in healthy male rats, the excretion of EVs into the urine appears to follow a circadian pattern [58]. This study reported that the highest excretion rate of EVs occurred during the active dark cycle (19,00–23,00). Additionally, the excretion rate of the EV associated protein TSG101 and the EV excretion rate showed a similar stoichiometry and circadian pattern. This finding suggest TSG101 can be used as a means of normalization of uEVs during time of day urine collections. The regulation of EV biogenesis and release in a circadian dependent manner for pathophysiological conditions is still currently under investigation. These studies are more complex since there are usually several variables associated with disease mechanisms that contribute to EV cargo loading and release.
