DNA in fresh urine supernatant is not affected by additional centrifugation and is protected against deoxyribonuclease

Abstract

Urinary DNA is widely studied as a non-invasive marker for monitoring of kidneys after transplantation or the progression of urinary tract tumors. The quantity of urinary DNA especially of mitochondrial origin has been reported to mirror kidney damage in various renal diseases and their models. Processing of samples might affect urinary DNA concentrations but the details are not clear. Samples of urine were collected from fifteen healthy volunteers. DNA was extracted from the whole urine, but also from the supernatant after centrifugation at 1600 g and 16000 g. In addition, we have analyzed the DNA in the microparticles in the pellet after the last spin. DNA was measured using fluorometry and real time PCR targeting nuclear and mitochondrial sequences. Addition of deoxyribonuclease to aliquots of samples enabled the characterization of DNA protection. Centrifugation at 1600 g decreased the concentration of extracted DNA by 66% at least in samples with higher DNA in whole urine. Interestingly, the additional spin at 16000 g did not result in a significant decrease in DNA concentration in the supernatant despite detectable microparticle-associated DNA. Deoxyribonuclease decreases total and nuclear DNA by 26% and 31% in whole urine. The majority of urinary mitochondrial DNA seems to be protected against deoxyribonuclease. Our results indicate high variability in urinary DNA even in healthy probands. Extracellular urinary DNA is partially bound to cell debris or microparticles, but a considerable part is still in the supernatant and is protected against cleavage. Further research should identify the nature of the protection, especially for mitochondrial DNA. Better understanding of the biology of urinary DNA should help its clinical interpretation.

Keywords: Cell-free DNA; DNase protection; Extracellular vesicles; Nephrology; Urinary markers.