Alessandro Rolfo

Chronic kidney disease may be differentially diagnosed from preeclampsia by serum biomarkers

Preeclampsia, affecting 5-8% of pregnancies, is the main cause of fetal-maternal mortality and morbidity. The differential diagnosis with chronic kidney disease (CKD) is a challenge owing to the overlapping clinical features. No biomarker has been found to discriminate between the two conditions. Here, we tested whether maternal serum levels of placental growth factor (PlGF) and soluble FMS-like tyrosine kinase-1 (sFlt-1), markers of preeclampsia, could be used to discriminate between 34 patients with preeclampsia, 23 patients with CKD during pregnancy, and 38 healthy pregnant women. Serum levels of PlGF and sFlt-1 were determined during the third trimester by commercially available immunoassays. In preeclampsia, sFlt-1 levels were significantly increased in comparison with that in CKD and in the control women. Serum levels of PlGF in preeclampsia were significantly decreased relative to both controls and patients with CKD. The sFlt-1 to PlGF ratio was significantly increased in preeclampsia (median 436) compared with controls (median 9.4) and CKD (median 4.0). No differences were found between controls and patients with CKD. Thus, our study suggests that it is possible to discriminate between preeclampsia and CKD during pregnancy by determining maternal serum levels of sFlt-1 and PlGF and their ratio.

Pre-eclampsia is associated with Helicobacter pylori seropositivity in Italy.

Objectives Pre-eclampsia (PE) is characterized by an excess of inflammation and endothelial dysfunction. Helicobacter pylori (H. pylori) causes chronic inflammatory changes and endothelial damage. We investigated the prevalence of seropositivity for IgG against H. pylori and cytotoxin-associated antigen A (CagA) in PE patients and the presence of H. pylori DNA in their placentas. Methods We tested 47 pregnant women with PE and 47 with uneventful pregnancies for serum antibodies against H. pylori (enzyme immunoassays) and CagA protein (immunoblot assays). In 20 of them (10 normal and 10 PE) we assessed the presence, in the placenta, of H. pylori DNA by means of nested polymerase chain reaction (PCR). The odds ratios (OR) and 95% confidence intervals (CI), adjusted for parity, were calculated using logistic regression analysis to assess the risk of PE associated with H. pylori infection. Results Helicobacter pylori seropositivity frequency was higher in mothers with PE (51.1%) compared to women with uneventful pregnancy (31.9%) (OR, 2.668; 95% CI, 1.084–6.566; P = 0.033). The difference was even greater for CagA seropositivity (80.9 and 14.9%, respectively) (OR, 26.035; 95% CI, 8.193–82.729; P < 0.001). All placentas were negative for H. pylori DNA. Conclusions Helicobacter pylori, and especially strains carrying the CagA gene, may contribute to the inflammatory mechanisms involved in the pathogenesis of PE.

Pro-inflammatory profile of preeclamptic placental mesenchymal stromal cells: new insights into the etiopathogenesis of preeclampsia.

The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF), Vascular Endothelial Growth Factor (VEGF), soluble FMS-like tyrosine kinase-1 (sFlt-1) and free β-human Chorionic Gonadotropin (βhCG) by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia. Methods Chorionic villous PDMSCs were isolated from severe preeclamptic (nu200a=u200a12) and physiological control term (nu200a=u200a12) placentae. Control and PE-PDMSCs’s cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM) was collected. Physiological villous explants (nu200a=u200a48) were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent. Results Cytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones. Conclusions Herein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs induced a PE-like phenotype in physiological villous explants. Our data clearly depict chorionic mesenchymal stromal cells as central players in placental physiopathology, thus opening to new intriguing perspectives for the treatment of human placental-related disorders as preeclampsia.

Pre-eclampsia or chronic kidney disease? The flow hypothesis

BACKGROUNDnChronic kidney disease (CKD) and pre-eclampsia (PE) occur in 3-5% of pregnancies. They often share hypertension and proteinuria and a differential diagnosis may be impossible. However, in PE, the pathogenesis is related to abnormal placentation, which can be detected by abnormal uterine and umbilical Doppler flow velocities, while in CKD, an intrinsic kidney disease is present. We hypothesize that Doppler studies can help to differentiate PE from CKD, as the flow velocities are altered in PE and normal in CKD.nnnMETHODSnWe retrospectively selected patients who were followed in our Materno-Foetal Unit (2005-10) and had at least one flow measurement in our setting. CKD patients were included in the presence of proteinuria (≥ 300 mg/day) and hypertension, mimicking PE. The clinical charts were reviewed by the same operators; the clinical diagnoses were taken as reference. Three flow patterns were considered: alteration of both flow velocity waveforms (FVWs) (uterine and umbilical arteries), hypothesized as predictive of PE; normal FVWs at both levels, hypothesized as predictive of CKD; altered FVW in either artery, considered mixed. Uterine FVWs were considered pathological according to the classical cut-point (RI > 0.58). Umbilical flows were evaluated according to standards adjusted for gestational age. Statistical analysis was performed in SPSS.nnnRESULTSnThe analysis included 61 cases. The presence of normal FVWs was significantly associated with the diagnosis of CKD (P = 0.0018). Conversely, the presence of both altered flows was significantly associated with PE (P = 0.0233).nnnCONCLUSIONSnIn the presence of proteinuria and hypertension, normal flows suggest CKD altered flows PE. Prospective studies are needed to refine this hypothesis based on the first Doppler criteria supporting the differential diagnosis between CKD and PE.

Association of Low-Protein Supplemented Diets with Fetal Growth in Pregnant Women with CKD

BACKGROUND AND OBJECTIVESnWomen affected by CKD increasingly choose to get pregnant. Experience with low-protein diets is limited. The aim of this study was to review results obtained from pregnant women with CKD on supplemented vegan-vegetarian low-protein diets.nnnDESIGN, SETTING, PARTICIPANTS, & MEASUREMENTSnThis was a single-arm, open intervention study between 2000-2012 of a low-protein diet in pregnant patients with stages 3-5 CKD or severe proteinuria (>1 g/d in the first trimester or nephrotic at any time). Stages 3-5 CKD patients who were not on low-protein diets for clinical, psychologic, or logistic reasons served as controls. The setting was the Obstetrics-Nephrology Unit dedicated to kidney diseases in pregnancy. The treated group included 24 pregnancies--21 singleton deliveries, 1 twin pregnancy, 1 abortion, and 1 miscarriage. Additionally, there were 21 controls (16 singleton deliveries, 5 miscarriages). The diet was a vegan-vegetarian low-protein diet (0.6-0.8 g/kg per day) with keto-acid supplementation and 1-3 protein-unrestricted meals allowed per week.nnnRESULTSnTreated patients and controls were comparable at baseline for median age (35 versus 34 years), referral week (7 versus 8), eGFR (59 versus 54 ml/min), and hypertension (43.5% versus 33.3%); median proteinuria was higher in patients on the low-protein diet (1.96 [0.1-6.3] versus 0.3 [0.1-2.0] g/d; P<0.001). No significant differences were observed in singletons with regard to gestational week (34 versus 36) or Caesarean sections (76.2% versus 50%). Kidney function at delivery was not different, but proteinuria was higher in the diet group. Incidence of small for gestational age babies was significantly lower in the diet group (3/21) versus controls (7/16; chi-squared test; P=0.05). Throughout follow-up (6 months to 10 years), hospitalization rates and prevalence of children below the third percentile were similar in both groups.nnnCONCLUSIONnVegan-vegetarian supplemented low-protein diets in pregnant women with stages 3-5 CKD may reduce the likelihood of small for gestational age babies without detrimental effects on kidney function or proteinuria in the mother.

Review: Feto-placental vascularization: A multifaceted approach

Doppler Ultrasound allows the in vivo study of feto-placental hemodynamics. Doppler flow velocity waveforms (FVWs) obtained from the umbilical arteries reflect downstream blood flow impedance, thus giving indirect evidence of vascular villous tree characteristics. Pulsatility Index, which quantifies FVWs, decreases throughout normal pregnancy, indicating decreasing impedance and is often higher in cases of fetal growth restriction (FGR). Different approaches (morphometrical, morphological, mathematical, immunohistochemical and molecular) have contributed to elucidation of which anomalies of the vascular villous tree underlie Doppler findings. 3D ultrasound may be useful in the study of feto-placental perfusion. However, the unsolved question is why developmental villous tree anomalies occur. Crucial to the success of future research is definition of the population studied based on the uniform and correct definition of FGR.

Nucleated Red Blood Cells in Term Fetuses: Reference Values Using an Automated Analyzer

Background: Nucleated red blood cells (NRBCs) are present in small numbers in the cord blood of healthy fetuses at birth. Acute and chronic stimuli, such as fetal hypoxia, cause increased circulating levels of NRBCs. Manual counting is presently the only way to quantify NRBCs, but it is time-consuming and inaccurate. Recently, automated approaches have been developed in order to quantify NRBCs. Objectives: Our aim was to compare manual vs. automated NRBC counting and derive reference values in umbilical cord blood samples from healthy term fetuses. Methods: We analyzed blood samples from 131 healthy term fetuses by the automated approach and calculated reference values of NRBC counts as percentiles. To compare automated NRBC count with manual count we obtained umbilical cord blood samples from 50 further fetuses. Results: Significant positive correlation was obtained between the two methods (r2 = 0.988, Bland-Altman plot mean difference NRBCs/100 WBC: –0.590). The median for NRBCs/100 WBC was 3.05 (range 0–11.6) and the median for absolute NRBC counts ×109/l was 0.39 (range 0–1.8). Conclusions: We demonstrate that values obtained with the automated NRBC counting method are comparable to those obtained with the microscopic manual evaluation and give reference values for umbilical cord NRBCs at term that can be used in clinical practice.

Is It Possible to Differentiate Chronic Kidney Disease and Preeclampsia by means of New and Old Biomarkers? A Prospective Study

Objective. Chronic kidney disease (CKD) and preeclampsia (PE) may both present with hypertension and proteinuria in pregnancy. Our objective is to test the possibility of distinguishing CKD from PE by means of uteroplacental flows and maternal circulating sFlt-1/PlGF ratio. Design. Prospective analysis. Population. Seventy-six patients (35 CKD, 24 PE, and 17 other hypertensive disorders), with at least one sFlt-1/PlGF and Doppler evaluation after the 20th gestational week. Methods. Maternal sFlt-1-PlGF were determined by immunoassays. Abnormal uterine artery Doppler was defined as resistance index ≥ 0.58. Umbilical Doppler was defined with gestational-age-adjusted Pulsatility Index. Clinical diagnosis was considered as reference. Performance of Doppler study was assessed by sensitivity analysis; sFlt-1/PlGF cut-off values were determined by ROC curves. Results. The lowest sFlt-1/PlGF ratio (8.29) was detected in CKD, the highest in PE (317.32) (P < 0.001). Uteroplacental flows were mostly preserved in CKD patients in contrast to PE (P < 0.001). ROC analysis suggested two cut-points: sFlt-1/PlGF ≥ 32.81 (sensitivity 82.93%; specificity 91.43%) and sFlt-1/PlGF ≥ 78.75 (sensitivity 62.89%, specificity 97.14%). Specificity reached 100% at sFlt-1/PlGF ≥ 142.21 (sensitivity: 48.8%). Early-preterm delivery was associated with higher sFlt-1/PlGF ratio and abnormal uteroplacental flows relative to late-preterm and term deliveries. Conclusions. sFlt-1/PlGF ratio and uteroplacental flows significantly correlated with PE or CKD and preterm delivery.

Pregnancy in Chronic Kidney Disease: questions and answers in a changing panorama.

Chronic kidney disease (CKD) is increasingly encountered in pregnancy because of greater diagnostic awareness, which is a reflection of the newer, broader definitions (i.e., any changes in blood or urine composition or at imaging, or a glomerular filtration rate (GFR) of <60xa0mL/min lasting at least 3 months) and of increased incidence (higher maternal age and better outcomes of several kidney diseases). CKD is extremely heterogeneous and may be described by the degree of GFR reduction (CKD stages), the presence of proteinuria and hypertension and the type of kidney disease; the risk of adverse pregnancy-related events increases as GFR decreases and it is affected by proteinuria and hypertension. Specific risks are reported in various diseases such as lupus nephropathy or diabetic nephropathy. While transplantation at least partially restores fertility in end-stage kidney disease, pregnancy on dialysis is increasingly reported. This chapter deals with the available evidence on the management of CKD patients in pregnancy.

Activating protein-1 family of transcription factors in the human placenta complicated by preeclampsia with and without fetal growth restriction

Preeclampsia (PE) is a serious disorder of human pregnancy, it is often associated with fetal growth restriction (FGR) which is a failure of the fetus to reach its own growth potential. Activator protein-1 (AP-1) is a family of transcription factors inducible in response to a variety of extracellular stimuli and functions. AP-1 plays a complex role in the regulation of different fundamental cellular processes, including cell proliferation, survival, death and transformation. We investigate the expression pattern of AP-1 transcription factors in normal placentas during gestation and in placentas from PE without and with FGR using semiquantitative RT-PCR and immunohistochemistry techniques. The most interesting data concern the alterations of protein expression patterns of c-fos, Jun D and c-jun in normal gestation as well as in PE and PE-FGR pathologies. In addition, alterations but not significant changes are detected in mRNA expressions for these transcription factors. We strongly suggest that c-fos is implicated in regulating invasiveness mechanism of extravillous trophoblast in normal gestation as well as in PE placentas. In addition, we suggest that the opposite modulation of Jun D and c-jun in PE and PE-FGR supports the recent hypothesis that PE and PE-FGR could be considered two pathologies with different origin (maternal and placental) each of which has a different molecular pattern of expression.