Urinary Extracellular Vesicles of Podocyte Origin and Renal Injury in Preeclampsia.

Renal histologic expression of the podocyte-specific protein, nephrin, but not podocin, is reduced in preeclamptic compared with normotensive pregnancies. We hypothesized that renal expression of podocyte-specific proteins would be reflected in urinary extracellular vesicles (EVs) of podocyte origin and accompanied by increased urinary soluble nephrin levels (nephrinuria) in preeclampsia. We further postulated that podocyte injury and attendant formation of EVs are related mechanistically to cellfree fetal hemoglobin (HbF) in maternal plasma. Our study population included preeclamptic (n=49) and normotensive (n=42) pregnant women recruited at delivery. Plasma measurements included HbF concentrations and concentrations of the endogenous chelators haptoglobin, hemopexin, and α1- microglobulin. We assessed concentrations of urinary EVs containing immunologically detectable podocyte-specific proteins by digital flow cytometry and measured nephrinuria by ELISA. The mechanistic role of HbF in podocyte injury was studied in pregnant rabbits. Compared with urine from women with normotensive pregnancies, urine from women with preeclamptic pregnancies contained a high ratio of podocin-positive to nephrin-positive urinary EVs (podocin+ EVs-to-nephrin+ EVs ratio) and increased nephrinuria, both of which correlated with proteinuria. Plasma levels of hemopexin, which were decreased in women with preeclampsia, negatively correlated with proteinuria, urinary podocin+ EVs-to-nephrin+ EVs ratio, and nephrinuria. Administration of HbF to pregnant rabbits increased the number of urinary EVs of podocyte origin. These findings provide evidence that urinary EVs are reflective of preeclampsia-related altered podocyte protein expression. Furthermore, renal injury in preeclampsia associated with an elevated urinary podocin+ EVs-to-nephrin+ EVs ratio and may be mediated by prolonged exposure to cellfree HbF.

First trimester prediction of preeclampsia.

Preeclampsia (PE) is a serious pregnancy-related condition that causes severe maternal and fetal morbidity and mortality. Within the recent years, there has been an increasing focus in predicting PE at the end of the first trimester of pregnancy. In this review, literature published between 2011 and 2015 was evaluated. In a total of six biomarker algorithms, for first and early second trimester, the prediction of preeclampsia is discussed. In addition, one randomized clinical trial was included. Several algorithms were based on placental biomarkers such as pregnancy-associated plasma protein A (PAPP-A), placental growth factor (PLGF), and soluble FMS-like tyrosine kinase 1 (s-FLT-1). The algorithms containing these biomarkers showed a high prediction rate (PR) for early onset PE, ranging from 44 to 92 % at 5 % false positive rate (FPR). New biomarkers suggest an alternative model based on free HbF and the heme scavenger alpha-1-microglobulin (A1M) with a prediction rate of 69 % at an FPR of 5 %. Interestingly, this model performs well without uterine artery Doppler pulsatility index (UtAD-PI), which is an advantage particularly if the screening method were to be implemented in developing countries. The randomized clinical trial showed a clear reduction in early onset PE as well as reducing preterm PE if identified high-risk pregnancies were treated with low-dose aspirin. In conclusion, PE prediction is now possible through several prediction algorithms and prophylaxis is beneficial in high-risk cases.

Fetal hemoglobin and α1-microglobulin as first- and early second-trimester predictive biomarkers for preeclampsia.

OBJECTIVE: The aim of this study was to evaluate fetal hemoglobin (HbF) and α(1)-microglobulin (A1M) in maternal serum as first-trimester biomarkers for preeclampsia (PE). STUDY DESIGN: The design was a case-control study. We included 96 patients in the first trimester of pregnancy (60 with PE and 36 controls). Venous serum samples were analyzed for HbF and total hemoglobin (Hb) by enzyme-linked immunosorbent assay and for A1M by radioimmunoassay. Sensitivity and specificity was calculated by logistic regression and receiver operating characteristic curve analysis. RESULTS: The HbF/Hb ratio and A1M concentration were significantly elevated in serum from women with subsequent development of PE (P < .0001). The optimal sensitivity and specificity was obtained using the biomarkers in combination; 69% sensitivity for a 5% screen positive rate and 90% sensitivity for a 23% screen positive rate. CONCLUSION: The study suggests that HbF/Hb ratio in combination with A1M is predictive biomarkers for PE.

Correction: The Human Endogenous Protection System against Cell-Free Hemoglobin and Heme Is Overwhelmed in Preeclampsia and Provides Potential Biomarkers and Clinical Indicators.

[This corrects the article DOI: 10.1371/journal.pone.0138111.].

The hemoglobin degradation pathway in patients with preeclampsia - Fetal hemoglobin, heme, heme oxygenase-1 and hemopexin - Potential diagnostic biomarkers?

OBJECTIVE: The aim of this study was to investigate how maternal cell-free fetal hemoglobin and heme impact the scavenger enzyme systems Hemopexin and Heme Oxygenase-1 in patients with preeclampsia (PE). The secondary aims were to evaluate these proteins as biomarkers for severity of the clinical manifestation i.e. hypertension, in early- and late onset PE. MATERIAL AND METHODS: Plasma samples taken within the last 24 h before delivery from 135 patients were analyzed, 89 PE and 46 normal pregnancies. All samples were analyzed for cell-free fetal hemoglobin (HbF), heme, hemopexin enzymatic activity (Hx activity), hemopexin concentration (Hx), and heme oxygenase 1 concentration (HO-1). Logistic regression analysis with ROC-curve analysis was performed to evaluate the possible use as biomarkers for preeclampsia. RESULTS: There were significantly higher levels of HbF (p = 0.01) and heme (0.01) but significantly lower Hx activity (p = 0.02), Hx (p < 0.0001) and HO-1 (p = 0.03) in PE plasma as compared to plasma of normal pregnancies. The Hx activity was significantly inversely correlated (p = 0.04) to the diastolic blood pressure. The HO-1 concentration was significantly inversely correlated to both the systolic and diastolic blood pressure (p = 0.01 and p = 0.003). ROC-curve analysis showed a combined detection rate for these biomarkers of 84% at 10% false positive rate. CONCLUSIONS: Increased maternal plasma levels of heme and HbF in PE are associated with decreased HO-1 and hemopexin protein levels as well as reduced hemopexin activity. By measuring the consumption of the scavenger protein Hx, and the proteins in the Hb degradation system, clinical information about the dynamics of the disease can be obtained.

Fetal hemoglobin, α1-microglobulin and hemopexin are potential predictive first trimester biomarkers for preeclampsia.

OBJECTIVE: Overproduction of cell-free fetal hemoglobin (HbF) in the preeclamptic placenta has been recently implicated as a new etiological factor of preeclampsia. In this study, maternal serum levels of HbF and the endogenous hemoglobin/heme scavenging systems were evaluated as predictive biomarkers for preeclampsia in combination with uterine artery Doppler ultrasound. STUDY DESIGN: Case-control study including 433 women in early pregnancy (mean 13.7weeks of gestation) of which 86 subsequently developed preeclampsia. The serum concentrations of HbF, total cell-free hemoglobin, hemopexin, haptoglobin and α1-microglobulin were measured in maternal serum. All patients were examined with uterine artery Doppler ultrasound. Logistic regression models were developed, which included the biomarkers, ultrasound indices, and maternal risk factors. RESULTS: There were significantly higher serum concentrations of HbF and α1-microglobulin and significantly lower serum concentrations of hemopexin in patients who later developed preeclampsia. The uterine artery Doppler ultrasound results showed significantly higher pulsatility index values in the preeclampsia group. The optimal prediction model was obtained by combining HbF, α1-microglobulin and hemopexin in combination with the maternal characteristics parity, diabetes and pre-pregnancy hypertension. The optimal sensitivity for all preeclampsia was 60% at 95% specificity. CONCLUSIONS: Overproduction of placentally derived HbF and depletion of hemoglobin/heme scavenging mechanisms are involved in the pathogenesis of preeclampsia. The combination of HbF and α1-microglobulin and/or hemopexin may serve as a prediction model for preeclampsia in combination with maternal risk factors and/or uterine artery Doppler ultrasound.

The Human Endogenous Protection System against Cell-Free Hemoglobin and Heme Is Overwhelmed in Preeclampsia and Provides Potential Biomarkers and Clinical Indicators.

Preeclampsia (PE) complicates 3-8% of all pregnancies and manifests clinically as hypertension and proteinuria in the second half of gestation. The pathogenesis of PE is not fully understood but recent studies have described the involvement of cell-free fetal hemoglobin (HbF). Hypothesizing that PE is associated with prolonged hemolysis we have studied the response of the cell-free Hb- and heme defense network. Thus, we have investigated the levels of cell-free HbF (both free, denoted HbF, and in complex with Hp, denoted Hp-HbF) as well as the major human endogenous Hb- and heme-scavenging systems: haptoglobin (Hp), hemopexin (Hpx), α1-microglobulin (A1M) and CD163 in plasma of PE women (n = 98) and women with normal pregnancies (n = 47) at term. A significant increase of the mean plasma HbF concentration was observed in women with PE. Plasma levels of Hp and Hpx were statistically significantly reduced, whereas the level of the extravascular heme- and radical scavenger A1M was significantly increased in plasma of women with PE. The Hpx levels significantly correlated with maternal blood pressure. Furthermore, HbF and the related scavenger proteins displayed a potential to be used as clinical biomarkers for more precise diagnosis of PE and are candidates as predictors of identifying pregnancies with increased risk of obstetrical complications. The results support that PE pathophysiology is associated with increased HbF-concentrations and an activation of the physiological Hb-heme defense systems.

PP006. Gene expression profiling of first trimester placentas from pregnancies at high risk of developing preeclampsia.

INTRODUCTION: Preeclampsia (PE) is an important cause of fetal and maternal morbidity and mortality. It is considered a two-stage disease, the first stage characterized by a defect placentation and the second stage by maternal manifestations. Details of the patho-physiology behind the transition from stage one to stage two remain unclear. OBJECTIVE: Was to study first trimester placental gene expression in patients identified as high risk for PE by either Doppler ultrasound or the biochemical markers cell free fetal hemoglobin (HbF) and alpha-1-microglobulin (A1M). METHODS: Placental samples were obtained from seven women at highrisk of PE as determined by Doppler ultrasound of the uterine arteries and eight women with normal uterine artery resistance who for other reasons terminated their pregnancies surgically. Maternal serum samples were analyzed for HbF and A1M. The patients were risk stratified according to two risk classifications: (I) High vs. low uterine artery resistance and (II) High HbF and A1M vs. low HbF and A1M. Total RNA from the placentas was used for whole genome microarray. The results were analyzed by bioinformatics and genes of interest confirmed with qPCR. RESULTS: A total of 453 and 332 significantly altered genes were identified in the two study groups. Bioinformatics revealed 12 genes of interest in study group I and 7 genes of interest in study group II. CONCLUSIONS: Genes related to vascular tonus regulation and inflammatory response were identified in study group I suggesting that a lack of tonus regulation and increased inflammation might contribute to the high uterine artery resistance seen in this group. Genes related to regulation of hematopoiesis was found in group II suggesting dysfunctional hematopoiesis as a factor explaining the high levels of cell-free HbF seen.