Janovičová Ľubica, Kmeťová Katarína, Tóthová Ľubomíra, Vlková Barbora, Celec Peter
Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia; Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
Mol Cell Probes. 2023 Apr;68:101900. doi: 10.1016/j.mcp.2023.101900. Epub 2023 Feb 18.
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.
尿DNA作为一种非侵入性标志物，被广泛用于监测肾移植后的肾脏情况或尿路肿瘤的进展。据报道，尿DNA的数量，尤其是线粒体来源的尿DNA数量，能够反映各种肾脏疾病及其模型中的肾损伤情况。样本处理可能会影响尿DNA浓度，但具体细节尚不清楚。我们从15名健康志愿者身上采集了尿液样本。DNA不仅从全尿中提取，也从1600g和16000g离心后的上清液中提取。此外，我们还分析了最后一次离心后沉淀中微粒的DNA。使用荧光法和针对核序列和线粒体序列的实时PCR测量DNA。向样本等分试样中添加脱氧核糖核酸酶能够对DNA保护进行表征。在全尿中DNA含量较高的样本中，1600g离心至少使提取的DNA浓度降低了66%。有趣的是，尽管可检测到与微粒相关的DNA，但16000g的额外离心并未导致上清液中DNA浓度显著降低。脱氧核糖核酸酶使全尿中的总DNA和核DNA分别降低了约26%和31%。大多数尿线粒体DNA似乎受到脱氧核糖核酸酶的保护。我们的结果表明，即使在健康受试对象中，尿DNA也存在高度变异性。细胞外尿DNA部分与细胞碎片或微粒结合，但相当一部分仍存在于上清液中，并受到切割保护。进一步的研究应确定这种保护的性质，尤其是对线粒体DNA的保护。更好地了解尿DNA的生物学特性将有助于其临床解读。
