Preeclampsia (PE) is a pregnancy complication affecting approximately 5% of women after 20 weeks’ gestation, characterized by new-onset hypertension and multi-organ dysfunction, particularly involving the kidneys. Early identification and management are crucial as PE significantly impacts maternal and fetal health. Previous studies have indicated challenges in diagnosing PE in patients with existing kidney conditions due to overlapping symptoms.
Cell-free DNA (cfDNA) has emerged as a potential biomarker across various medical contexts, reflecting organ injury levels. Specifically, kidney-specific cell-free DNA (ks-cfDNA) may provide valuable insight into renal injury during conditions like PE.
This exploratory study aims to assess the feasibility of quantifying urinary ks-cfDNA and to determine whether levels significantly differ between women diagnosed with PE and matched normotensive pregnant controls.
This was a single-centre, prospective case-control study conducted at Mercy Hospital for Women, Heidelberg, Australia, from March to October 2023. The study recruited 20 women with PE and 20 normotensive pregnant (NP) controls, both groups matched for gestational age. All participants provided written informed consent, following approval from the Mercy Health Human Research Ethics Committee.
PE was diagnosed according to the Society of Obstetric Medicine of Australia and New Zealand (SOMANZ) criteria, and women were excluded from the NP group if they had pre-existing hypertension, chronic kidney disease, or fetal growth restrictions.
Urine samples were collected and analyzed using digital PCR to quantify three kidney-specific epigenetic targets (ks-et): ks-et1 (pan-kidney marker), ks-et2 (renal proximal tubular epithelial cell and podocyte marker), and ks-et3 (specific RPTEC marker). These were compared against total cfDNA levels normalized using the beta-actin gene.
Using methods developed in previous studies, researchers established kidney-specific epigenetic markers derived from DNA methylation signatures. The identified markers include ks-et1 (PAX2), ks-et2 (GRAMD1B), and ks-et3 (DDC). Each marker serves as a potential indicator of kidney health and is explored through their presence in urine samples from participants.
Total cfDNA quantification was performed using the beta-actin gene as a control for assessing overall cfDNA levels. The quantifications facilitated a comparative analysis between urinary ks-cfDNA and total cfDNA levels, and sensitivities of individual markers were evaluated.
Participants supplied a mid-stream urine sample, which underwent immediate cooling and centrifugation to reduce contamination. Following extraction and bisulfite conversion, the samples were analyzed using digital PCR to determine concentrations of ks-cfDNA.
Urine creatinine levels were also measured, given their significance in assessing kidney function. Statistical analyses, including Mann-Whitney U tests and Receiver Operating Characteristic (ROC) curves, were utilized to evaluate and compare the biomarker performance across groups.
The analysis revealed that urinary levels of ks-cfDNA markers ks-et2 and ks-et3 were significantly higher in the PE cohort. Specifically, median concentrations were 231 copies/mL for ks-et2 and 81 copies/mL for ks-et3 in the PE group, compared to 62 copies/mL and 20 copies/mL in NP controls, respectively. However, no significant differences for ks-et1 and total cfDNA were observed.
Further, urinary creatinine levels were also elevated in women with PE (10.9 mmol/L vs. 3.9 mmol/L in NP), highlighting its potential as a reliable indicator of PE with an AUC of 0.76, outperforming ks-cfDNA markers.
These findings indicate that the quantification of urinary ks-cfDNA is feasible and shows promise as an indirect measure of renal injury associated with preeclampsia. While urinary creatinine remains a strong predictor of PE, the study opens avenues for future research into the clinical implications of ks-cfDNA as a potential biomarker for early detection and monitoring of kidney injury in pregnant populations.
In conclusion, this study supports the hypothesis that levels of urinary ks-cfDNA are elevated in women with preeclampsia, indicating kidney injury. However, further investigations are warranted to establish the clinical utility of ks-cfDNA in diagnosing and managing preeclampsia. The work encourages future studies exploring the predictive abilities of these biomarkers in broader contexts of pregnancy-related complications.