Vegan-vegetarian low-protein supplemented diets in pregnant CKD patients: fifteen years of experience.

BACKGROUND: Pregnancy in women with advanced CKD becoming increasingly common. However, experience with low-protein diets in CKD patients in pregnancy is still limited. Aim of this study is to review the results obtained over the last 15 years with moderately restricted low-protein diets in pregnant CKD women (combining: CKD stages 3-5, proteinuria: nephrotic at any time, or > =1 g/24 at start or referral; nephrotic in previous pregnancy). CKD patients on unrestricted diets were employed for comparison. STUDY PERIOD: January, 2000 to September, 2015: 36 on-diet pregnancies (31 singleton deliveries, 3 twin deliveries, 1 pregnancy termination, 1 miscarriage); 47 controls (42 singleton deliveries, 5 miscarriages). The diet is basically vegan; since occasional milk and yoghurt are allowed, we defined it vegan-vegetarian; protein intake (0.6-0.8 g/Kg/day), keto-acid supplementation, protein-unrestricted meals (1-3/week) are prescribed according to CKD stage and nutritional status. Statistical analysis was performed as implemented on SPSS. RESULTS: Patients and controls were similar (p: ns) at baseline with regard to age (33 vs 33.5), referral week (7 vs 9), kidney function (CKD 3-5: 48.4 % vs 64.3 %); prevalence of hypertension (51.6 % vs 40.5 %) and proteinuria >3 g/24 h (16.1 % vs 12.2 %). There were more diabetic nephropathies in on-diet patients (on diet: 31.0 % vs controls 5.3 %; p 0.007 (Fisher)) while lupus nephropathies were non-significantly higher in controls (on diet: 10.3 % vs controls 23.7 %; p 0.28 (Fisher)). The incidence of preterm delivery was similar (<37 weeks: on-diet singletons 77.4 %; controls: 71.4 %). The incidence of other adverse pregnancy related outcomes was non-significantly lower in on-diet patients (early preterm delivery: on diet: 32.3 % vs controls 35.7 %; birth-weight = <1.500 g: on diet: 9.7 % vs controls 23.8 %). None of the singletons in the on-diet series died, while two perinatal deaths occurred among the controls (p = 0.505). The incidence of small for gestational age (SGA <10th centile) and/or extremely preterm babies (<28th week) was significantly lower in singletons from on-diet mothers than in controls (on diet: 12.9 % vs controls: 33.3 %; p: 0.04 (Fisher)). CONCLUSION: Moderate protein restriction in the context of a vegan-vegetarian supplemented diet is confirmed as a safe option in the management of pregnant CKD patients.

Macrophage migration inhibitory factor in fetoplacental tissues from preeclamptic pregnancies with or without fetal growth restriction.

The proinflammatory cytokine MIF (macrophage migration inhibitory factor) is involved in physiological and pathological processes in pregnancy. MIF maternal serum levels are increased in preeclampsia (PE). We hypothesize that pregnancy tissues are the source of MIF overexpression in PE. MIF protein was studied in maternal sera, placental tissues, fetal membranes, and umbilical cord of 8 control and 20 PE pregnancies: 10 with normal fetal growth (PE-AGA) and 10 with fetal growth restriction (PE-FGR). MIF levels were significantly higher in PE-AGA membranes than in controls and PE-FGR. In PE-FGR, MIF cord concentrations were higher than in PE-AGA while MIF placental levels were lower than in controls. MIF maternal serum levels were higher in PE, compared to controls, and the difference was mainly due to PE-FGR samples. These data support MIF involvement in PE pathogenesis and suggest that different pregnancy tissues contribute to MIF production in PE with and without fetoplacental compromise.

Vegetarian supplemented low-protein diets. A safe option for pregnant CKD patients: report of 12 pregnancies in 11 patients.

BACKGROUND: Pregnancy in CKD is an increasing challenge, considering also the paucity of therapeutic tools available in pregnant women. While theoretically interesting, the experience with low protein diets in pregnancy is limited. Aim of this feasibility study is to review our experience with supplemented vegetarian low protein diets in pregnancy, as a "rescue treatment" for severe CKD and/or proteinuria. METHODS: Data were gathered prospectively. Diet schema: proteins: 0.6-0.7 g/Kg/day, amino and chetoacid supplementation, 1-3 free meals/week. Compliance, side effects, biochemical data recorded at each visit (at least twice monthly). RESULTS: Between January 2000 and February 2010, out of 168 pregnancies referred, 12 were managed by the diet (11 patients; median age 33, range 20-38). One pregnancy was terminated (patient's choice); the other 10 patients delivered 11 healthy babies. At referral, 2 patients were in stage 4 CKD, 4 in stage 3, 4 had nephrotic proteinuria (3.6-6.3 g/day). One patient doubled serum creatinine; none needed renal replacement therapy within 6 months from delivery. No patient complained of side effects, nor developed hyperkalemia or hypercalcaemia. Two babies from mothers in CKD stage 4 were small for gestational age; 9/11 were delivered by caesarean section (median gestational age 33 weeks: range 28-37; birth weight 935-2620 g) within a policy of delivery in the presence of foetal growth impairment and/or worsening of proteinuria, GFR, hypertension or foetal conditions. All babies are well, 1 month, 7.5 years from delivery. CONCLUSION: Our report suggests considering vegetarian diets as an additional tool in the management of pregnant CKD patients.

Placental expression of soluble fms-like tyrosine kinase 1 is increased in singletons and twin pregnancies with intrauterine growth restriction.

OBJECTIVE: Intrauterine growth restriction (IUGR) is characterized by decreased placental perfusion. Low oxygen has been shown to increase soluble fms-like tyrosine kinase 1 (sFlt-1) expression in the human placenta. The objective of this study was to examine sFlt-1 expression in different types of IUGR pregnancies, including early-onset severe cases characterized by abnormal umbilical and uterine artery Doppler and discordant IUGR twins in which the normal cotwin represents the optimal control because both placentas share the same uterine environment. PATIENTS: Placentas from four subgroups were collected: early severe IUGR with umbilical artery absent end diastolic flow (n = 19), small for gestational age with normal uterine and umbilical artery Doppler (n = 11), severely growth-restricted dichorionic and monochorionic twins with abnormal umbilical artery Doppler (n = 9), preeclamptic twins (n = 3), and age-matched normal singletons (n = 19) and twin controls (n = 8). RESULTS: Expression of sFlt-1 mRNA and protein was significantly increased in IUGR placentas compared with small for gestational age and normal control placentas. sFlt-1 expression levels were also significantly greater in the small IUGR twin placentas from discordant twin pregnancies compared with the normal cotwin. In preeclamptic twins, sFlt-1 expression was increased in only one of the two placentas. CONCLUSIONS: Our results demonstrate that sFlt-1 expression is increased in severe IUGR placentas with abnormal umbilical artery Doppler of singletons and also in discordant IUGR twins. Reduced placental perfusion may contribute to the increased expression of sFlt-1 in IUGR pregnancies. Our data are compatible with differential sFlt-1 expression in placentas from discordant twins.

Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise.

Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16INK4A, p18INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16INK4A, p18INK4C, CDK4 and CDK6 expressions in physiological term villous explants. PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16INK4A, p18INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay. We reported significantly increased p16INK4A and p18INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16INK4 and p18INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast. We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise.

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.

Increased levels of macrophage migration inhibitory factor (MIF) in preeclampsia.

OBJECTIVE: MIF is a proinflammatory cytokine involved in reproduction. Systemic activation of maternal inflammatory cell responses may play an important role in the pathogenesis of preeclampsia (PE). We hypothesized that MIF could be involved in preeclampsia. STUDY DESIGN: Concentration of immunoreactive MIF was assayed by enzyme-linked immunoassorbent assay (ELISA) in maternal serum samples obtained from 41 term control pregnancies and 21 severe preeclamptic pregnancies (14 delivered before and 7 at or after 34 weeks). RESULTS: MIF serum levels were significantly higher in preeclamptic pregnancies (median 12.74 ng/ml) than in control group (median 5.3n g/ml) p = 0.001. MIF concentration was significantly higher when delivery occurred <34 weeks (median 17.80 ng/ml; range 2.80-80.20) than in the group delivered > or = 34 weeks (median 6.16 ng/ml; range 1.62-23.65) p = 0.037. CONCLUSIONS: High maternal serum levels MIF in pregnancies complicated by severe preeclampsia strongly support the role of inflammation in the pathogenesis of this disease.

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

UNLABELLED: 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 ß-human Chorionic Gonadotropin (ßhCG) 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 (n = 12) and physiological control term (n = 12) 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 (n = 48) 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 ßhCG 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 ßhCG 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.

Central nervous system microangioarchitecture in the human foetus.

It is thought that arterioles penetrating the central nervous system behave as terminal arteries and lack for anastomosys. The purpose of our study was to define the angiogenesys in the fetal encephalon at different stages of development. To this purpose, we examinated 13 fetal and newborn encephalons between the 10th and 33rd week. To label blood vessels, we used an immunohistochemical procedure based on the detection of two antigens located within endothelial cells: CD31 and CD34. The cerebral vascularization modifies in quantity and in structure during pregnancy, with important topographic differences between cerebral cortex and striatal-limbic areas. We observed two microarchitectural patterns: 1. Rectangular mesh pattern, characterized by capillaries that join transversally to one or more branches that deepen orthogonally from the surface of the meninges; 2. Hexagonal mesh pattern, which surrounds small groups of neurons and develops with a honeycomb shape. The rectangular mesh pattern is mostly observed from the 13th to 26th week in the white matter, in the hippocampus and in the cortex. The hexagonal mesh pattern is typical of the basal nuclei, and of the cerebral cortex during the 10th-12th week and after the 26th-27th week. Until the 26th week the vascularization increases mainly in the hippocampus and in the basal nuclei. The cortex shows a vascularization increment, greater than in the limbic system, with a pattern prevalently hexagonal in areas were the neurons' number increases. Our data demonstrate that, in the human fetus, cerebral capillaries are not of terminal type. On the contrary, they show a rich anastomotic network that has different patterns in white matter (rectangular pattern) or in grey matter (hexagonal pattern). The functional meaning of this difference is unknown, but we can suppose that its role is to warrant availability of nutritional substances within regions where a high number of neurons is present. Recent findings in computational neuroanatomy show that computer simulated axonall symmetric bifurcation can generate a dendritic tree with close similarities with real observed vascular patterns in fetal cortex.