**4. Regulation of size distribution**

The size of EVs may or may not depend on the amount and type of cargo enriched in the vesicles. Some studies have shown an increase in EV release without a change in EV size, while other studies have shown the contrary [17, 18]. Apoptotic bodies represent the largest type of EVs and contain organelles, in addition to nucleic acids, proteins, and lipids. Conversely, exosomes and microvesicles lack organelles, but contain specific molecules of interest.

Numerous studies have shown the same cell type can produce subpopulations of exosomes that are remarkable different in size. For example, polarized epithelial cells, such as those in the kidney tubule release exosomes across the apical plasma membrane and the basolateral plasma membrane (**Figure 2**). These two types of exosomes show distinct differences in their size and protein composition [19]. Interestingly, Matsui et al. showed two independent mechanisms for exosome release across the apical and basolateral plasma membranes [20]. This group showed that ceramide plays an essential role in basolateral exosome release whereas, ALIX is important for apical exosome release [20].

Although it is reasonable to expect the size of EVs to be directly proportional to the concentration of a particular molecule present in the EVs, this is not always the case. For example, Chacko et al. showed urinary EV concentration decreased after infusing Tempol in hypertensive 129Sv mice while the size of the EVs increased [21].

#### **Figure 2.**

*Release of EV's from various kidney cell types. All cell types in the kidney including glomerular cells and cells within each segment of the nephron release EV's that can allow for intercellular communication, intracellular signaling, and the regulation of various mechanisms in health and disease. Polarized epithelial cells of the nephron release two distinct populations of EV's across the apical membrane that faces the lumen and the basolateral membrane that faces the peritubular capillaries. EV's that are released into the lumen containing the filtrate can be taken up by recipient cells downstream in the nephron or excreted from the body in the form of urine. EV's that are released across the basolateral membrane can be reabsorbed back into the blood.*

The amount of the EV marker protein annexin A2, but not the amount of other EV marker proteins including TSG101 or flotillin-2 decreased after tempol treatment [21].
