223 Summary and general discussion Advancing urine-based molecular tests using alternative urine biomarkers • Innovative sequencing platforms allow the simultaneous analysis of genetic and epigenetic features, including mutations, copy number alterations, and changes in (hydroxy)methylation. • Alternative urine biomarkers with high potential include cfDNA fragmentation profiles, histone levels and post-translational histone modifications, nucleosome positioning, circulating RNA, circulating proteins, circulating metabolites, and exfoliated tumor cells. 8.5 Unraveling the origin of urine cell-free DNA The urgency to implement liquid biopsy tests in the clinic may distract attention away from understanding the origin of cfDNA. Yet, enhancing our understanding of cfDNA biology and the origin of urine cfDNA is essential to improve its future clinical utility (49, 118, 119). During the last decades, several hypotheses on the glomerular filtration of cfDNA have been proposed, including passive filtration through pores or active vesicle-mediated transport (120, 121). 8.5.1 Transrenal transport of cell-free DNA by passive filtration Passive filtration may occur for the clearing of short cfDNA fragments through the glomerular filtration barrier (120). However, circulating cfDNA fragments in both blood and urine are typically histone-bound (85) with a molecular weight of around 200 kilodaltons (kDa) (122). Under normal conditions, proteins with a molecular weight above 70-80 kDa are hindered from passing through the glomerular filtration barrier due to their size (123). The glomerular membrane permeability reduces further for negatively charged molecules because of the negatively charged characteristics of the glomerular basement membrane itself (124). Alternatively, the concept of protein filtration through larger shunt-like pores has been described (125). However, the prevalence of such pores is either extremely low or they may even be nonexistent according to the most recent literature (126). Given this information, it is unlikely that nucleosomes pass through the glomerular membrane in their typical configuration. In the bloodstream, nucleosomes are protected from nuclease digestion and rendered soluble due to the substitution of histone H1 with the serum amyloid P component (SAP) (127). Although hypothetical, it is possible that binding to SAP mediates nucleosome unwinding which might in turn facilitate glomerular passing (121). An intriguing finding is that the tissue amyloid P component is naturally present on the glomerular basement membrane, supporting its potential role in nucleosome clearance (128). 8
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