Oxygen regulation of aquaporin-4 in human placenta.

RESEARCH QUESTION: We recently reported that blocking of placental aquaporins (AQP) abrogates the apoptotic response of the trophoblast. As trophoblast apoptosis is exacerbated in pre-eclampsia, we hypothesized that changes in AQP in these placentae may trigger programmed cell death. We analysed AQP4 expression in pre-eclamptic placentae and its regulation by oxygen tension. DESIGN: AQP4 expression was studied in placentae from non-pathological and pre-eclamptic pregnancies by reverse transcription polymerase chain reaction (RT-PCR), Western blot, immunofluorescence and immunohistochemistry. Explants from non-pathological placentae were cultured in normoxia, hypoxia, hypoxia-reoxygenation and CoCl2. AQP4 expression was investigated by RT-PCR and Western blot. Hypoxia responsive elements sites on AQP4 promotor were investigated by in-silico analysis. AQP4 degradation was studied in the presence of proteosomal and lysosomal inhibitors. RESULTS: AQP4 protein expression was weakly detectable in pre-eclamptic placentae, but its mRNA was elevated compared with non-pathological placentae. In non-pathological explants cultured in hypoxia, AQP4 mRNA and protein were increased compared with placentae cultured in ambient oxygen but decreased after reoxygenation. Incubation with CoCl2, that stabilizes hypoxia inducible factor (HIF)-1α, also increased AQP4 levels. In-silico analysis showed three putative binding sites for HIF-1α in AQP4 promotor. CONCLUSIONS: Oxygen may regulate AQP4 expression in human placenta, possibly through HIF-1α. Therefore, the decrease in AQP4 throughout pregnancy, previously reported, is consistent with changes in HIF-1α, and suggests that AQP4 might have a crucial role during placentation. Therefore, the abnormal expression of AQP4 may be involved in the cause of pre-eclampsia, but it does not seem to take part in the apoptotic events.

New Insights Into the Role of Placental Aquaporins and the Pathogenesis of Preeclampsia.

Accumulated evidence suggests that an abnormal placentation and an altered expression of a variety of trophoblast transporters are associated to preeclampsia. In this regard, an abnormal expression of AQP3 and AQP9 was reported in these placentas. Recent data suggests that placental AQPs are not only water channel proteins and that may participate in relevant processes required for a normal placental development, such as cell migration and apoptosis. Recently we reported that a normal expression of AQP3 is required for the migration of extravillous trophoblast (EVT) cells. Thus, alterations in this protein might lead to an insufficient transformation of the maternal spiral arteries resulting in fluctuations of oxygen tension, a potent stimulus for oxidative damage and trophoblast apoptosis. In this context, the increase of oxygen and nitrogen reactive species could nitrate AQP9, producing the accumulation of a non-functional protein affecting the survival of the villous trophoblast (VT). This may trigger the exacerbated release of apoptotic VT fragments into maternal circulation producing the systemic endothelial dysfunction underlying the maternal syndrome. Therefore, our hypothesis is that the alteration in the expression of placental AQPs observed at the end of gestation may take place during the trophoblast stem cell differentiation, disturbing both EVT and VT cells development, or during the VT differentiation and turnover. In both situations, VT is affected and at last the maternal vascular system is activated leading to the clinical manifestations of preeclampsia.

Systemic effects of Subtilase cytotoxin produced by Escherichia coli O113:H21.

Subtilase cytotoxin (SubAB) is a member of the AB5 cytotoxin family and is produced by certain strains of Shiga toxigenic Escherichia coli. The toxin is known to be lethal to mice, but the pathological mechanisms that contribute to Uremic Hemolytic Syndrome (HUS) are poorly understood. In this study we show that intraperitoneal injection of a sublethal dose of SubAB in rats triggers a systemic response, with ascitic fluid accumulation, heart hypertrophy and damage to the liver, colon and kidney. SubAB treated rats presented microalbuminuria 20 days post inoculation. At this time we found disruption of the glomerular filtration barrier and alteration of the protein reabsorption mechanisms of the proximal tubule. In the kidney, SubAB also triggered an epithelial to mesenchymal transition (Wuyts et al., 1996). These findings indicate that apart from direct cytotoxic effects on renal tissues, SubAB causes significant damage to the other organs, with potential consequences for HUS pathogenesis. IMPORTANCE: Uremic Hemolytic Syndrome is an endemic disease in Argentina, with over 400 hundred new cases each year. We have previously described renal effects of Shiga Toxin and its ability to alter renal protein handling. Bearing in mind that Subtilase Cytotoxin is an emerging pathogenic factor, that it is not routinely searched for in patients with HUS, and that to the date its systemic effects have not been fully clarified we decided to study both its systemic effects, and its renal effects to assess whether SubAB could be contributing to pathology seen in children.

Brain antioxidant responses to acute iron and copper intoxications in rats.

Dose- and time-dependent antioxidant responses to Fe (0-60 mg kg(-1)) and Cu overloads (0-30 mg kg(-1)) in rat brains are described by the C50 and the t1/2, the brain metal concentration and the time for half maximal oxidative responses. Brain GSH and the GSH/GSSG ratio markedly decreased after Fe and Cu treatments (50-80%) with a t1/2 of 9-10 h for GSH and of 4 h for GSH/GSSG for both metals. The GSH/GSSG ratio was the most sensitive indicator of brain oxidative stress. The decrease of GSH and the increase of in vivo chemiluminescence had similar time courses. The C50 for brain chemiluminescence, GSH and hydrophilic and lipophilic antioxidants were in similar ranges (32-36 µg Fe g(-1) brain and 10-18 µg Cu g(-1) brain), which indicated a unique free-radical mediated process for each metal. The brain concentration of hydrophilic and lipophilic antioxidants decreased after Fe and Cu loads; hydrophilic antioxidants decreased by 46-68% with a t1/2 of 10-11 h and lipophilic antioxidants decreased by 75-45% with a t1/2 of 10-12 h. Cu,Zn-SOD and CAT activities and the protein expression were adaptively increased (100-90% after Fe and Cu loads), with a t1/2 of 8-12 h. GPx-4 activity decreased after both metal loads by 73-27% with a t1/2 of 8-4 h with decreased protein expression.

Rat liver antioxidant response to iron and copper overloads.

The rat liver antioxidant response to Fe and Cu overloads (0-60mg/kg) was studied. Dose- and time-responses were determined and summarized by t1/2 and C50, the time and the liver metal content for half maximal oxidative responses. Liver GSH (reduced glutathione) and GSSG (glutathione disulfide) were determined. The GSH content and the GSH/GSSG ratio markedly decreased after Fe (58-66%) and Cu (79-81%) loads, with t1/2 of 4.0 and 2.0h. The C50 were in a similar range for all the indicators (110-124µgFe/g and 40-50µgCu/g) and suggest a unique free-radical mediated process. Hydrophilic antioxidants markedly decreased after Fe and Cu (60-75%; t1/2: 4.5 and 4.0h). Lipophilic antioxidants were also decreased (30-92%; t1/2: 7.0 and 5.5h) after Fe and Cu. Superoxide dismutase (SOD) activities (Cu,Zn-SOD and Mn-SOD) and protein expression were adaptively increased after metal overloads (Cu,Zn-SOD: t1/2: 8-8.5h and Mn-SOD: t1/2: 8.5-8.0h). Catalase activity was increased after Fe (65%; t1/2: 8.5h) and decreased after Cu (26%; t1/2: 8.0h), whereas catalase expression was increased after Fe and decreased after Cu overloads. Glutathione peroxidase activity decreased after metal loads by 22-39% with a t1/2 of 4.5h and with unchanged protein expression. GSH is the main and fastest responder antioxidant in Fe and Cu overloads. The results indicate that thiol (SH) content and antioxidant enzyme activities are central to the antioxidant defense in the oxidative stress and damage after Fe and Cu overloads.

High levels of human chorionic gonadotropin (hCG) correlate with increased aquaporin-9 (AQP9) expression in explants from human preeclamptic placenta.

Trophoblastic abnormalities have a central role in the pathophysiology of preeclampsia, and some placental hormones, such as human chorionic gonadotropin (hCG), could affect the placental function. Here, we hypothesized that the elevated serum levels of hCG may be involved in the increased aquaporin-9 (AQP9) protein expression in preeclamptic placentas via adenosine 3('),5(')-cyclic phosphate (cAMP) pathways. Normal placental explants were cultured with different concentrations of recombinant hCG or 8-Br-cAMP, a potent analogue of cAMP. We evaluated AQP9 protein expression and localization. After both treatments, we localized AQP9 in the apical membrane of syncytiotrophoblast and in the cytoplasm. We also observed a concentration-dependent effect on AQP9 protein expression. In addition, water uptake increased 1.6-fold in explants treated with hCG. Our results suggest that hCG may increase AQP9 protein expression and functionality via cAMP pathways. Although, in preeclamptic placentas high levels of hCG may upregulate AQP9 protein expression, AQP9 functionality was reduced possibly by other factors.

Efficient immune responses against Intimin and EspB of enterohaemorragic Escherichia coli after intranasal vaccination using the TLR2/6 agonist MALP-2 as adjuvant.

Mucosal vaccine formulations based on purified recombinant C280 gamma-Intimin and EspB (Escherichia coli secreted protein B) from enterohaemorragic E. coli co-administered with a pegylated derivative of the TLR2/6 agonist MALP-2 (macrophage-activating lipopeptide) as adjuvant were evaluated in BALB/c mice. After intranasal vaccination, strong humoral and cellular immune responses were observed against C280 gamma-Intimin and EspB. Sera of immunized mice inhibit bacterial haemolytic activity in vitro. Antigen-specific T-cell proliferation, IL-4, IL-2 and IFN-gamma producing cells, and secretory IgA were mostly detected in animals receiving MALP-2 as adjuvant. These results suggest that C280 gamma-Intimin and EspB are good candidate antigens to be incorporated into mucosal vaccines against this important pathogen.