Proteomic profiling of urine identifies specific fragments of SERPINA1 and albumin as biomarkers of preeclampsia

Abstract

Objective: The cause of preeclampsia remains unknown and the diagnosis can be uncertain. We used proteomic-based analysis of urine to improve disease classification and extend the pathophysiologic understanding of preeclampsia.

Study design: Urine samples from 284 women were analyzed by surface-enhanced laser desorption/ionization. In the exploratory phase, 59 samples were used to extract the proteomic fingerprint characteristic of severe preeclampsia requiring mandated delivery and to develop a diagnostic algorithm. In the challenge phase, we sought to prospectively validate the algorithm in 225 women screened for a variety of high- and low-risk conditions, including preeclampsia. Of these, 19 women were followed longitudinally throughout pregnancy. The presence of biomarkers was interpreted relative to clinical classification, need for delivery, and other urine laboratory measures (ratios of protein to creatinine and soluble fms-like tyrosine kinase-1 to placental growth factor). In the translational phase, biomarker identification by tandem mass spectrometry and validation experiments in urine, serum, and placenta were used to identify, quantify, and localize the biomarkers or related proteins.

Results: We report that women with preeclampsia appear to present a unique urine proteomic fingerprint that predicts preeclampsia in need of mandated delivery with highest accuracy. This characteristic proteomic profile also has the ability to distinguish preeclampsia from other hypertensive or proteinuric disorders in pregnancy. Pregnant women followed longitudinally who developed preeclampsia displayed abnormal urinary profiles more than 10 weeks before clinical manifestation. Tandem mass spectrometry and de novo sequencing identified the biomarkers as nonrandom cleavage products of SERPINA1 and albumin. Of these, the 21 amino acid C-terminus fragment of SERPINA1 was highly associated with severe forms of preeclampsia requiring early delivery. In preeclampsia, increased and aberrant SERPINA1 immunoreactivity was found in urine, serum, and placenta, in which it localized predominantly to placental villi and placental vascular spaces adherent to the endothelium. In addition, significant perivascular deposits of misfolded SERPINA1 aggregates were exclusively identified in preeclamptic placentae.

Conclusion: Proteomics-based characterization of urine in preeclampsia identified a proteomic fingerprint composed of SERPINA1 and albumin fragments, which can accurately diagnose preeclampsia and shows promise to discriminate it from other hypertensive proteinuric diseases. These findings provide insight into a novel pathophysiological mechanism of preeclampsia related to SERPINA1 misfolding, which may offer new therapeutic opportunities in the future.

Figures

Figure 1. Flow-chart and distribution of urine samples for the challenge phase

Pregnancy-associated conditions unrelated to preeclampsia were preterm labor: n=47; preterm premature rupture of the membranes: n=11, placenta praevia: n=4, sepsis: n=1 and isolated IUGR: n=1. Of the women enrolled in the cross-sectional cohort, 86 had a medically-indicated delivery for preeclampsia. The remaining 110 pregnant women delivered at different gestational ages. Deliveries at <37 weeks in this group occurred either spontaneous (n=50) or were medically-indicated for clinical conditions such as: placenta previa (n=4), non-reassuring fetal status (n=10) or idiopathic intra-uterine fetal demise (n=2). Out of the 19 women enrolled in the longitudinal cohort of the challenge phase, 3 developed sPE or spPE requiring mandated delivery at <37 weeks. Abbreviations: PE: preeclampsia; sPE: severe preeclampsia; mPE: mild preeclampsia; crHTN: chronic hypertension; CRL: healthy pregnant control; wks: weeks.

Figure 2. The urinary proteomic fingerprint of severe preeclampsia requiring delivery

Representative SELDI profile from a patient with severe preeclampsia (sPE) at 30 weeks gestational age illustrating the 13 biomarker components (P1–P13, marked with red bullets) of the urinary proteomic scores (UPSb=13 and UPSr=47) presented in comparison with a SELDI profile of urine from a healthy pregnant control patient (both UPSb and UPSr of 0) at the same gestational age (CRL). kDa: kilodaltons. The asterisk marks a complex of peaks not part of the UPS profile that accompanies P1–P3 complex.

Figure 3. Diagnostic performances of the proteomic profile relative to other analytes assessed in the same random urine sample

Receiver operating characteristic (ROC) curves of the proteomic score UPSr (red line), protein-to-creatinine ratio (blue line) and soluble fms-like tyrosine kinase 1 [sFlt-1]-to-placental growth factor [PlGF] ratio (green line) for predicting a preeclampsia-related indicated delivery. Analysis included all consecutive pregnant women who contributed with urine samples to the cross-sectional challenge phase (n=196).

Figure 4. Evolution of urinary proteomics scores during pregnancy in the longitudinal cohort of the challenge phase

19 asymptomatic pregnant women at low or high risk for developing preeclampsia were followed longitudinally from the first trimester until 6 weeks postpartum. UPSb (Panel A) and UPSr (Panel B) scores were generated prospectively. Black boxes represent the women who developed preeclampsia requiring an mandated preterm delivery (n=3). Open boxes represent women who had a normal course of their pregnancy (n=16). On the x axis are the time periods in weeks prior to each patient’s delivery date (time 0). pp = postpartum. On the y axis are the values of the urinary proteomic scores (UPSb and UPSr, respectively). The arrow indicates the time-point where women manifested clinical signs and symptoms. The data is presented as percentiles with median. The ends of the boxes define the 25th and 75th percentiles, the line inside the box defines the median and the whiskers show the largest and smallest values. 2-way ANOVA: p<0.01 for both time periods and outcome. Bars with at least one common red letter are not statistically different at a value of p>0.05.

Figure 5. Quantitative and qualitative changes in SERPINA-1 immunoreactivity in urine and serum of women with severe preeclampsia

Fractional excretion (FE) of SERPINA-1 (proportion of SERPINA-1 immunoreactivity excreted in the urine compared with that filtered by the glomeruli) relative to that of total proteins (Panel A) and albumin (Panel B), in logarithmic format. Despite the significant urinary loss, circulating SERPINA-1 levels are increased in women with severe preeclampsia (sPE) (n=29) compared to controls (CRL, n=16) at similar gestational ages (sPE: 32.7±4.4 weeks vs. CRL: 30.4±1.5 weeks, p=0.151) (Panel C). Western blot of SERPINA-1 immunoreactivity in urine from representative CRL and sPE women under denaturing (Panel D) or native conditions (Panel E). The red arrow points to monomeric SERPINA-1 (S1: 52 kDa). The bands above 52 kDa represent supramolecular aggregates while those below represent fragments. These results suggest that women with sPE excrete a complex pattern of SERPINA-1 fragments and aggregates in addition to P1–P3 biomarkers identified by us using SELDI. Lane 2 in panel E was loaded with SERPINA-1 polymerized in vitro (pS1) to demonstrate antibody reactivity against oligomeric SERPINA-1. Molecular weight markers (M) in kilodaltons (kDa) are loaded in Lane 1.

Figure 6. Immunohistochemistry of SERPINA-1 and of its supramolecular aggregates in human placenta

Representative placental sections from a woman that delivered after a medically-indicated preterm delivery for severe preeclampsia (Panels a–;d & g) and a gestational age matched patient with spontaneous preterm birth (panels e–;g) were stained immunohistochemically with a polyclonal antibody recognizing the multiple immunoreactive forms (Panels a–;c & e–;f) and with a monoclonal antibody (ATZ11) recognizing only the supramolecular aggregates of SERPINA-1 (oligomers, misfolded forms and SERPINA-1-elastase complexes). Positive staining is shown by Ni-DAB (purple-black) deposits. PVS: placental villous stroma; PTR: placental trophoblast; PBV: placental blood vessels; Endo: endothelium. Panel g: sPE: omitted primary antibody. Magnification scales are presented for each panel in microns. Panels b, c, f & g: light methyl-green counterstain.