224 Chapter 8 8.5.2 Transrenal transport of cell-free DNA by extracellular vesicles Another likely scenario of transrenal passing of cfDNA is through extracellular vesicles (EVs), of which exosomes are most intensively studied. EVs play a key role in intercellular communication and are involved in a wide variety of physiological and pathological processes, including cancer (129). The lipid bilayer surrounding EVs acts as a protective layer against nucleases and immune cells (130). It has been described that EVs carry nucleic acids, including cfDNA (131). However, the precise localization of nucleic acids in EVs is still under debate (i.e. enclosed within the lumen or surface-bound) (118, 132). Localization might also be affected by physiological factors, as it has been shown that exercise specifically increased exosomal DNA levels on their surface (133). The average fragment size of exosome-derived DNA appeared longer as compared to the classical size of cfDNA, with fragment sizes up to 4000 base pairs (134, 135). This could explain the presence of long DNA fragments in the urine supernatant fraction (90). Urine comprises different types of EVs, which are mostly derived from the urinary tract but may also originate from more distant anatomical sites (136). It is currently unknown how EVs are transported from the blood to the urine. Exosomes, which are amongst the smallest extracellular vesicles, have a size range from 40 to 100 nm. Therefore, translocation through glomerular pores seems unlikely, as only vesicles with a size range of 6 to 8 nm can pass (137). In a diseased state, disruptions of the membrane pores might occur which could potentially facilitate the passing of larger vesicles (138). Alternatively, vesicles could enter the urine by being absorbed by the proximal tubule cells or podocytes via selective endocytosis (139, 140). 8.5.3 Tubular reabsorption of cell-free DNA after glomerular filtration After glomerular filtration, tubular reabsorption occurs in which various substances are transported back into the bloodstream to regulate the urine composition (141). The potential reabsorption of cfDNA after filtration might be a crucial determinant of total cfDNA levels in the urine. The biological functions of cfDNA molecules, including their immunogenic effects and role in cellular homeostasis (49, 118), provide reasons for potential reabsorption after filtration. Moreover, efficient recycling of circulating nucleotides could conserve energy and resources within the body and help to maintain a balance between DNA synthesis and degradation. From an energy-conserving perspective, it is most likely that the reabsorption of cfDNA predominantly occurs via passive mechanisms. Presently, this area remains largely underexplored and warrants deeper investigation.
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