225 Summary and general discussion 8.5.4 Experimental approaches to study transrenal excretion of cell-free DNA Further studies are required to elucidate the precise mechanisms involved in the transrenal transport of cfDNA. This process could be studied by modeling kidney filtration systems in vitro and exposing such systems to cfDNA released by cultured cancer cells. We are currently in the process of initiating such pilot experiments. So far, we have observed that cfDNA can pass through a monolayer of human renal proximal tubular epithelial cells in a transwell setting, indicating that cfDNA is potentially reabsorbed into the bloodstream after glomerular filtration. Previous studies have also shown the value of in vitro cell culture models to study the release mechanisms of cfDNA (47, 142). Over the years, innovative models to mimic the kidney system have become available, such as a glomerulus-on-a-chip, three-dimensional microfluidic models, and kidney organoids. However, despite recent progress, mimicking the glomerular filtration barrier or proximal tubule remains challenging due to the physiologically complex and specialized nature of these kidney structures (143). Nevertheless, combining currently available kidney models with novel sequencing platforms to characterize excreted cfDNA in detail could offer a powerful approach to advance our understanding of how nucleic acids enter the urine. A more in-depth understanding of transrenal clearance of cfDNA could guide the optimization of sampling procedures and ensure that urinebased tests yield clinically meaningful results, which both represent vital factors for the successful translation of urine-based tests into clinical practice (118). Unraveling the origin of urine cell-free DNA • Understanding mechanisms underlying transrenal clearance of cfDNA is crucial for successful urine test development. • Potential mechanisms of glomerular filtration include passive filtration through pores or active vesicle-mediated transport. • Reabsorption of cfDNA after glomerular filtration could influence total cfDNA levels in the urine. • The development of in vitro models mimicking transrenal transport of cfDNA would be valuable to elucidate transrenal transport mechanisms. 8
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