Introduction
Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by endothelial dysfunction, renal impairment, and systemic inflammation. It affects 2-8% of pregnancies and remains a major cause of maternal and perinatal morbidity and mortality worldwide [1]. Despite advances in antenatal surveillance, reliable biomarkers for early diagnosis and risk stratification are lacking.
Netrin-1 is an axonal guidance molecule that plays a role in angiogenesis and vascular function and has been implicated in endothelial injury and inflammation. Kidney injury molecule-1 (KIM-1) is a sensitive marker of renal tubular damage. Both have been suggested as potential biomarkers for PE; however, the data remain limited and inconsistent [2,3].
This study aimed to assess serum Netrin-1 and urinary KIM-1 levels in preeclamptic and normotensive pregnant women and to evaluate their diagnostic and prognostic performance.
Materials and methods
1. Study design
This prospective observational study was conducted at the Departments of Obstetrics and Gynecology and Biochemistry at a tertiary care referral center in Western Rajasthan from September 2023 to March 2025. Ethical approval was obtained from the Institutional Ethics Committee.
2. Study population
Pregnant women aged >18 years diagnosed with preeclampsia (American College of Obstetricians and Gynecologists [ACOG] 2020) were included. The controls were healthy, normotensive pregnant women without proteinuria or obstetric complications. Women who were already on medication to manage PE before enrollment, had a urinary tract infection, were of advanced maternal age (>40 years), or had a previous history of stroke or subarachnoid hemorrhage, chronic hypertension, renal disease, diabetes, autoimmune disorders, multiple gestations, or other systemic illnesses were excluded.
3. Sample size
Based on previous literature [4], 102 participants were needed to detect a significant difference in serum Netrin-1 between groups (power 80%; α=0.05). After accounting for a 15% attrition rate, 118 women (59 patients and 59 controls) were enrolled.
4. Data collection and assays
Data, including demographic details, clinical parameters, and relevant laboratory investigations, were collected from each participant. All enrolled participants were followed up on until delivery. Maternal complications such as eclampsia, hemolysis (red blood cell breakdown), elevated liver enzymes, and low platelet count (HELLP) syndrome, placental abruption, renal failure, hepatic failure, and pulmonary edema, as well as fetal complications including preterm birth, fetal growth restriction (FGR), low birth weight, and stillbirth, were recorded.
5. Diagnostic criteria for PE
According to the ACOG 2020, PE is diagnosed as hypertension (systolic ≥140 mmHg or diastolic ≥90 mmHg on two occasions at least 4 hours apart) that occurs after 20 weeks of gestation and is accompanied by proteinuria: ≥300 mg/24-hour urine OR urine protein/creatinine ratio ≥0.3 OR urine dipstick ≥2+ OR.
Any of the following signs indicate systemic involvement: platelets <100,000/μL, serum creatinine >1.1 mg/dL, aspartate aminotransferase and alanine aminotransferase, pulmonary edema, headache, or visual disturbances.
During the study period, PE and gestational hypertension were managed according to international standard guidelines.
Blood and urine samples were collected from both the PE group (cases) and healthy pregnant women (controls). Venous blood (10 mL) was drawn from each participant for routine laboratory investigations and measurement of serum Netrin-1 levels. Urine samples were collected to assess urinary KIM-1 levels and proteinuria. Proteinuria was evaluated using both the dipstick method and 24-hour urinary protein estimation. Serum Netrin-1 and urinary KIM-1 levels were quantified using enzyme-linked immunosorbent assay techniques.
6. Outcomes
The primary outcomes were differences in serum Netrin-1 and urinary KIM-1 levels between the groups. Secondary outcomes included associations with the severity of PE, maternal complications (e.g., eclampsia, HELLP syndrome, and abruption), and fetal outcomes (small for gestational age [SGA], FGR, stillbirth, and preterm birth).
7. Statistical analysis
Data were analyzed using SPSS version 30.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as mean±standard deviation and median (interquartile range) values and categorical variables as numbers (percentages). Student’s t-test was used to compare normally distributed data, the Mann-Whitney U-test was used to compare non-normally distributed data, and Pearson’s chi-square or Fisher’s exact test was used to compare categorical variables. Receiver operating characteristic (ROC) curve analysis was performed to determine diagnostic accuracy. The area under the curve (AUC) and optimal cut-off values were calculated. The optimal cut-off value was calculated using the Youden index. Statistical significance was set at P<0.05 for all comparisons.
Results
1. Characteristics of study participants
This study included 118 women (59 with PE and 59 controls). The mean age was 28.22±5.2 years for the PE group and 27.22±3.93 years for the control group. Baseline characteristics were comparable, except for higher urea, creatinine, lactate dehydrogenase, and proteinuria levels in patients with PE (Table 1).
2. Biomarker levels
Both biomarker levels were higher in the PE group than in the control group (Netrin-1: 844.55±195.27 vs. 378.24±127.75 pg/mL; P<0.0001; KIM-1: 1.57±0.75 vs. 0.62±0.29 ng/mg creatinine; P<0.0001).
3. Comparison of serum Netrin-1 and urinary KIM-1 levels stratified by severity of PE
Women with PE with severe features exhibited significantly higher serum Netrin-1 and urinary KIM-1 levels than those without severe features (Netrin-1: 889.85±208.25 vs. 762.59±139.27 pg/mL; P=0.020; KIM-1: 1.86±0.75 vs. 1.06±0.40 ng/mg creatinine; P<0.0001).
4. Predictive value of Netrin-1 and KIM-1 for maternal and fetal complications in PE
The markedly higher levels of these biomarkers (Netrin-1: 1,001.35±190.49 vs. 757.90±135.98 pg/mL; KIM-1: 2.29±0.56 vs. 1.18±0.51 ng/mg creatinine), with P<0.0001, suggest a strong association between elevated biomarker levels and the risk of maternal complications. No significant differences were found in biomarker levels for SGA, FGR, stillbirth, or preterm birth.
6. Comparative diagnostic performance of serum Netrin-1 and urinary KIM-1 in early- and late-onset PE
Serum Netrin-1 levels demonstrated excellent diagnostic accuracy for both early- and late-onset PE. For early-onset cases, it showed an AUC of 0.993 with a cut-off >614.02 pg/mL, identifying 92% of affected women and achieving 100% specificity. The positive predictive value (PPV) was 100% and the negative predictive value (NPV) was 89.47%. In late-onset PE, serum Netrin-1 performed similarly well with an AUC of 0.993 at a cut-off >617.33 pg/mL, correctly identifying 94.12% of cases, with 100% specificity, a PPV of 100%, and an improved NPV of 95.45%. Urinary KIM-1 also shows strong diagnostic capability, but with some variation between early- and late-onset PE. In early-onset PE, urinary KIM-1 had an AUC of 0.948 and a cut-off >1.27 ng/mg creatinine, detecting 76% of cases with 100% specificity, a PPV of 100%, and an NPV of 73.91%. For late-onset PE, urinary KIM-1 demonstrated an AUC of 0.897 at a cut-off >0.67 ng/mg creatinine, identifying 97.06% of cases but with lower specificity (73.81%) and PPV (75%). However, it maintained a high NPV of 96.88%, indicating strong reliability in ruling out late-onset disease when negative (Figs. 2, 3).
Discussion
PE is one of the most common hypertensive complications of pregnancy. Systemic intravascular inflammation, endothelial cell dysfunction, increased thrombin production, an anti-angiogenic state, and multiple organ involvement are the hallmarks of PE [5].
PE has been linked to inadequate maternal spiral artery remodeling. In PE, defective trophoblast invasion occurs during the first few weeks of pregnancy, resulting in poor placental perfusion in the second half of pregnancy [6]. Pro-angiogenic and anti-angiogenic factors play a significant role in the trophoblastic invasion process during vascular remodeling. This balance is disrupted by increased anti-angiogenic factor production, which leads to systemic endothelial dysfunction, manifesting as PE. The developing placenta establishes several pro-angiogenic (vascular endothelial growth factor, placenta-induced growth factor [PIGF]) and anti-angiogenic (soluble feline McDonough sarcoma, such as soluble fms-like tyrosine kinase-1 [sFlt-1]) factors, and it has been recognized that a balance among these factors is necessary for normal placental development [7].
This study examined the clinical, biochemical, and biomarker profiles of preeclamptic pregnancies and evaluated the diagnostic and prognostic relevance of serum Netrin-1 and urinary KIM-1 levels.
This study demonstrated that serum Netrin-1 and urinary KIM-1 levels were significantly elevated in preeclamptic women compared with those in healthy pregnant controls. Both biomarkers were correlated with disease severity and maternal complications, supporting their role in risk stratification.
Serum Netrin-1 showed excellent diagnostic performance, with near-perfect sensitivity and specificity exceeding those of previously studied markers, such as the sFlt-1/PIGF ratio [8].
Our findings align with earlier studies suggesting a role for Netrin-1 in vascular homeostasis and endothelial dysfunction in PE [4,9-11]. Urinary KIM-1 levels were also significantly higher in the PE group (P<0.0001). These findings are consistent with those of other studies, confirming its usefulness as a non-invasive marker of renal tubular injury, a hallmark of PE. Urinary KIM-1 levels were significantly elevated in early-onset cases (P=0.003), suggesting that renal dysfunction becomes more pronounced when PE occurs during the early stages of pregnancy.
A similar significant increase was observed in PE patients with severe features (P<0.0001), implying that the severity of renal dysfunction is proportional to the severity of the disease and emphasizing the use of urinary KIM-1 in renal stress monitoring [12-16]. However, neither biomarker predicted fetal complications, highlighting the multifactorial etiology of adverse perinatal outcomes. As per the study, serum Netrin-1 and urinary KIM-1 are promising biomarkers for the diagnosis and severity stratification of PE. Serum Netrin-1, in particular, demonstrates excellent diagnostic accuracy and may have potential utility in clinical risk prediction models. Further multicenter and longitudinal studies are required to validate these findings.
Our findings are comparable to those reported by Berenji et al. [11], who demonstrated the diagnostic potential of serum Netrin-1 and urinary KIM-1 levels in PE. Our study expands on this work in several important ways. First, we evaluated both early- and late-onset PE, demonstrating that the diagnostic performance of Netrin-1 remains strong across subtypes, whereas the previous study focused primarily on early-onset disease. Second, we examined associations with maternal complications and found significantly higher biomarker levels in women who developed severe complications, such as HELLP syndrome and renal impairment-an aspect not addressed in the earlier study. Finally, ROC analysis revealed higher diagnostic accuracy for Netrin-1 (AUC, 0.993), suggesting stronger clinical predictive value. These distinctions highlight the broader clinical utility and risk stratification potential of these biomarkers in diverse PE presentations.
Biologically, the elevation of Netrin-1 and KIM-1 in PE can be explained by their roles in vascular and renal injury pathways. Netrin-1 is normally expressed in endothelial cells and contributes to vascular integrity through anti-inflammatory and pro-angiogenic signaling. In PE, placental hypoxia and oxidative stress lead to endothelial dysfunction, prompting compensatory upregulation of Netrin-1 in an attempt to restore vascular stability. Conversely, KIM-1 is highly upregulated in proximal renal tubular epithelial cells in response to ischemic or inflammatory injury. Its elevation in PE likely reflects renal tubular damage secondary to glomerular endotheliosis and reduced renal perfusion. Thus, increased levels of both biomarkers reflect parallel endothelial and renal injury processes, supporting their utility in assessing disease severity.
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