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Hephaestin (Hp) is a trans-membrane protein, which plays a critical role in intestinal iron absorption. Hp was originally identified as the gene responsible for the phenotype of sex-linked anaemia in the sla mouse. The mutation in the sla protein causes ac-cumulation of dietary iron in duodenal cells, causing severe microcytic hypochromic anaemia. Although mucosal uptake of dietary iron is normal, export from the duodenum is inhibited. Hp is homologous to ceruloplasmin (Cp), a member of the family of multi copper fer-roxidases (MCFs) and possesses ferroxidase activity that facilitates iron release from the duodenum and load onto the serum iron trans-port protein transferrin. In the present study, attempts were made to produce biologically active recombinant mouse hephaestin as a secretory form tagged with green fluorescent protein (GFP), Hpsec-GFP. Plasmid expressing Hpsec-GFP was constructed and transfected into COS and CHO cells. The GFP aided the monitoring expression in real time to select the best conditions to maximise expression and provided a tag for purifying and analysing Hpsec-GFP. The protein had detectable oxidase activity as shown by in-gel and solution-based assays. The methods described here can provide the basis for further work to probe the interaction of hephaestin with other proteins using complementary fluorescent tags on target proteins that would facilitate the fluorescence resonance energy transfer measurements, for example with transferrin or colocalisation studies, and help to discover more about hephaestin works at the molecular level.

Purpose There is general agreement that some dietary polyphenols block non-haem iron uptake, but the mechanisms by which they achieve this action are poorly understood. Since the polyphenol quercetin is ingested daily in significant amounts, we have investigated the effect of quercetin on duodenal non-haem iron absorption in vivo, as well as its effect on factors known to be involved in systemic iron metabolism. Methods Rats were subject to gastric gavage and systemic quercetin administration. Treatments were followed with uptake studies using radiolabeled iron, serum iron and transferrin saturation measurements, LC-MS/MS analysis of quercetin metabolites in serum, determination of tissue non-haem iron content and analysis of gene expression of iron-related proteins. Results Both oral and intraperitoneal (IP) quercetin caused serum and tissue iron depletion by two means, first by increasing mucosal iron uptake and inhibiting iron efflux from duodenal mucosa, and second by decreasing levels of duodenal DMT1, Dcytb and FPN. Additionally, IP quercetin induced highly significant increased liver expression of hepcidin, a hormone known to inhibit intestinal iron uptake. Conclusions Oral quercetin significantly inhibited iron absorption, while IP quercetin significantly affected iron-related genes. These results could lead to development of new effective ways of preventing and treating iron deficiency anaemia, the most widespread nutritional disorder in the world. Electronic supplementary material The online version of this article (10.1007/s00394-018-1680-7) contains supplementary material, which is available to authorized users.

Iron metabolism during pregnancy is biased toward maintaining the fetal supply, even at the cost of anemia in the mother. The mechanisms regulating this are not well understood. Here, we examine iron deficiency and supplementation on the hierarchy of iron supply and the gene expression of proteins that regulate iron metabolism in the rat. Dams were fed iron-deficient diets for 4 wk, mated, and either continued on the deficient diet or an iron-supplemented diet during either the first half or the second half of their pregnancy. A control group was maintained on normal iron throughout. They were killed at 0.5, 12.5, or 21.5 days of gestation, and tissues and blood samples were collected. Deficiency and supplementation had differential effects on maternal and fetal hematocrit and liver iron levels. From early in pregnancy, a hierarchy of iron supply is established benefiting the fetus to the detriment of the mother. Transferrin receptor, transferrin receptor 2, and hepcidin mRNA expression were regulated by both iron deficiency and supplementation. Expression patterns showed both organ and supplementation protocol dependence. Further analysis indicated that iron levels in the fetal, and not maternal, liver regulate the expression of liver transferrin receptor and hepcidin expression in the mother.

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 ± 0.5% of the total dose was transported after 8 h, equivalent to 38.8 ± 2.1 pmol·cm−2·h−1. Transfer of Tf across the cell layer was much more limited; 2.4 ± 0.2% of the total dose was transported after 8 h, equivalent to 5.0 ± 0.4 pmol·cm−2·h−1. Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta.

The liver expresses batteries of cytoprotective genes that confer cellular resistance to oxidative stress and xenobiotic toxins, and protection against cancer and other stress-related diseases. These genes are mainly regulated by Nrf2, making this transcription factor a target for small molecule discovery to treat such diseases. In this report, we identified dietary polyphenolic antioxidants that not only activated these genes but also relieved Nrf2 repression by Keap1, a Cul3-dependent ubiquitin ligase adaptor protein that mediates its degradation. Analysis of postprandial liver RNA revealed a marked activation of both genes by all test polyphenols compared with controls. Nrf2 inhibition by RNA interference reduced polyphenol effects on its target gene expression. Our data suggest that polyphenols may induce cellular defense genes by derepressing Nrf2 inhibition by Keap1. We posit that this ability to derepress Nrf2 and reactivate its target genes may underlie the protection conferred by polyphenols against oxidative stress-related diseases.

We investigated the potential of a new family of lipidic peptide dendrimers in protein transduction into cultured cells. Dendrimer-protein interaction was determined by gel retardation assays using purified recombinant protein. To assess intracellular protein delivery, two marker proteins were used: recombinant firefly luciferase and a Cy3-labeled monoclonal antibody to the c-myc proto-oncogene. Protein delivery was determined by luciferase assays and fluorescence microscopy, respectively. While there was minimal delivery of luciferase or antibody in the absence of the dendrimers, the latter increased protein delivery substantially. Luciferase delivery was concentration and cell type-dependent; the efficiency of delivery also varied with the number of terminal amino groups on the dendrimers. In previous reports, we showed that these dendrimers could be used for gene and drug delivery; the data we report herein suggest that they may also be capable of intracellular protein delivery. This finding has important implications for the use of these dendrimers in protein therapeutics and vaccinology.

Glomerular expression of the ATP-sensitive P2X7 receptor in diabetic and hypertensive rat models.BackgroundThe molecular identification and characterization of the adenosine triphosphate (ATP)-sensitive family of P2 receptors is comparatively new. There are two main subgroups, each with several subtypes and widespread tissue distribution, including the kidney. A unique member of the P2X subgroup of P2 receptors is the ATP-gated ion channel P2X7, which on activation can cause cell blebbing, cytokine release, and cell death by necrosis or apoptosis. We report expression of this receptor in normal rat kidney and in two chronic models of glomerular injury: streptozotocin-induced (STZ) diabetes and ren-2 transgenic (TGR) hypertension.MethodsAt different time points in these models, we used a polyclonal antibody to the P2X7 receptor and immunohistochemistry to determine its expression and distribution. We also used Western blotting and real-time polymerase chain reaction (PCR) to detect changes in P2X7 receptor protein and mRNA expression, respectively.ResultsWe found only low-level glomerular immuno-staining for the P2X7 receptor in normal rat kidney, but intense P2X7 receptor immunostaining of glomeruli in kidneys from diabetic animals at 6 and 9 weeks, and in hypertensive animals at 12 weeks. In diabetic animals, real-time PCR demonstrated a ∼tenfold increase in glomerular P2X7 receptor mRNA relative to control, and Western blotting confirmed an increase in protein. Immunohistochemistry and immunoelectron microscopy showed staining of glomerular podocytes, which was both intracellular and at the plasma membrane.ConclusionWe conclude that the P2X7 receptor is not expressed appreciably under normal conditions, but that following glomerular injury it is significantly up-regulated, mainly in podocytes, though also in endothelial and mesangial cells, of animals with STZ-induced diabetes mellitus or TGR hypertension. Although the exact function and regulation of this receptor remain unclear, its association with inflammatory cytokine release and cell death suggests that increased expression might be involved in the pathogenesis of glomerular cell injury or repair.

Iron (Fe) deficiency anaemia during pregnancy results in an increased risk of perinatal mortality and morbidity and is a significant factor for increased risk of disease in later life. Consequently we have developed a rat model to study the relationship between maternal Fe deficiency and postnatal growth and blood pressure in the offspring. Weanlings were fed a control or Fe-deficient diet prior to and throughout pregnancy. At term, all pups were cross-fostered to control fed dams and weaned onto control diet. At birth, pups from deficient dams had a greater mortality rate, were smaller and had reduced haematocrit and liver Fe levels. They also had larger hearts, smaller kidneys and spleens and unchanged livers (relative organ weight). The pups grew normally. At 6 weeks, male pups from deficient dams had a higher and females a lower blood pressure than their normal counterparts. At 10 and 16 weeks, blood pressure in the males from deficient dams was still raised and in the females was now greater than controls. The haematocrit was lower in males throughout the 16 weeks and in females until 10 weeks of age. There was no significant difference in the offsprings' liver Fe stores at 6, 10 or 16 weeks. Duodenal Fe uptake in both the Fe-deficient mother and newborn offspring was significantly increased. By cross-fostering, we have eliminated confounding factors, such as maternal anaemia during lactation and show, unequivocally, that prenatal nutrition is critical for the development of normal postnatal function.

Hephaestin was implicated in mammalian iron homeostasis following its identification as the defective gene in murine sex-linked anaemia. It is a member of the family of copper oxidases that includes mammalian ceruloplasmin, factors V and VIII, yeast fet3 and fet5 and bacterial ascorbate oxidase. Hephaestin is different from ceruloplasmin, a soluble ferroxidase, in having a membrane-spanning region towards the C-terminus. Here we report the gene structure, spanning approximately 100 kb, of the human homologue of mouse hephaestin. The sequence was assembled from the cDNA clones and the chromosome X genomic sequence data available at the Sanger Centre. It has an open reading frame that encodes a protein of 1158 residues, 85% identical with the murine homologue. A model of the N-terminal ecto-domain has been built based on the known three-dimensional structure of human ceruloplasmin. The overall tertiary structure for the hephaestin and the putative residues involved in binding copper and iron appear to be highly conserved between these proteins, which suggests they share the same fold and a conserved function.

Maternal iron deficiency during pregnancy induces anaemia in the developing fetus; however, the severity tends to be less than in the mother. The mechanism underlying this resistance has not been determined. We have measured placental expression of proteins involved in iron transfer in pregnant rats given diets with decreasing levels of iron and examined the effect of iron deficiency on iron transfer across BeWo cell layers, a model for placental iron transfer. Transferrin receptor expression was increased at both mRNA and protein levels. Similarly, expression of the iron-responsive element (IRE)-regulated form of the divalent metal transporter 1 (DMT1) was also increased. In contrast, the non-IRE regulated isoform showed no change in mRNA levels. Protein levels of DMT1 increased significantly. Iron efflux is thought to be mediated by the metal transporter protein, IREG1/ferroportin1/MTP1, and oxidation of Fe(II) to Fe(III) prior to incorporation into fetal transferrin is carried out by the placental copper oxidase. Expression of IREG1 was not altered by iron deficiency, whereas copper oxidase activity was increased. In BeWo cells made iron deficient by treatment with desferrioxamine (‘deferioxamine’), iron accumulation from iron-transferrin increased, in parallel with increased expression of the transferrin receptor. At the same time, iron efflux also increased, showing a higher flux of iron from the apical to the basolateral side. The data show that expression of placental proteins of iron transport are up-regulated in maternal iron deficiency, resulting in an increased efficiency of iron flux and a consequent minimization of the severity of fetal anaemia.

There is considerable current interest in the cytoprotective effects of natural antioxidants against oxidative stress. In particular, epicatechin, a major member of the flavanol family of polyphenols with powerful antioxidant properties in vitro, has been investigated to determine its ability to attenuate oxidative-stress-induced cell damage and to understand the mechanism of its protective action. We have induced oxidative stress in cultured human fibroblasts using hydrogen peroxide and examined the cellular responses in the form of mitochondrial function, cell-membrane damage, annexin-V binding and caspase-3 activation. Since one of the major metabolites of epicatechin in vivo is 3′-O-methyl epicatechin, we have compared its protective effects with that of epicatechin. The results provide the first evidence that 3′-O-methyl epicatechin inhibits cell death induced by hydrogen peroxide and that the mechanism involves suppression of caspase-3 activity as a marker for apoptosis. Furthermore, the protection elicited by 3′-O-methyl epicatechin is not significantly different from that of epicatechin, suggesting that hydrogen-donating antioxidant activity is not the primary mechanism of protection.

Axial distribution and characterization of basolateral P2Y receptors along the rat renal tubule. Background Several groups have identified P2Y receptors in the basolateral membrane of the rat nephron. These studies have not covered all segments of the nephron and have relied solely on the relative potency of receptor agonists for classification. Methods We measured purine and pyrimidine-induced changes in intracellular free calcium concentration ([Ca 2+ ] i ) in anatomically defined segments of the rat nephron. To complement these functional studies, we have used reverse transcription-polymerase chain reaction methodology to identify specific P2Y receptor transcripts in these segments. Results Adenosine 5′-triphosphate (ATP) mobilized [Ca 2+ ] i in all nephron segments, except for the thick ascending limb of Henle, which was poorly responsive. Adenosine (100 μmol/L) was without effect, confirming that the effect of ATP was mediated by P2 receptors. In the proximal convoluted tubule (PCT) and outer medullary collecting duct (OMCD), there was evidence for two receptor subtypes with characteristics of P2Y 1 - and either P2Y 2 - or P2Y 4 -like receptors. A novel finding in the thin limbs was the presence of a receptor with properties of both P2Y 2 and P2Y 4 receptor subtypes. To aid classification, we identified P2Y receptor mRNA in rat nephron segments. In the PCT and OMCD and thin ascending limb of Henle, we found expression of P2Y 1 , P2Y 2 , and P2Y 4 receptors. In the descending limb of Henle, P2Y 1 and P2Y 2 mRNA was found, but P2Y 4 was not expressed. Conclusion These data suggest that extracellular ATP can influence tubular cell function in all segments of the rat nephron, through P2Y receptors via multiple (and coexpressed) P2Y receptor subtypes.

Journal URL: http://cancerres.aacrjournals.org/

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Journal URL: http://carcin.oxfordjournals.org/

P2Y receptors couple to G proteins and either mobilize intracellular Ca2+or alter cAMP levels to modulate the activity of Ca2+- and cAMP-sensitive ion channels. We hypothesize that increased ion transport into the lumen of MDCK cysts can osmotically drive fluid movement and increase cyst size. Furthermore, activation of the adenylate cyclase/cAMP pathway may trigger cell proliferation via an extracellular signal-related kinase cascade. To test this hypothesis, several P2Y receptor inhibitors were used on the MDCK in vitro model of renal cyst formation. The nonspecific P2 receptor inhibitors reactive blue 2 and suramin reduced cyst growth significantly, as did PPADS and, to a lesser extent, the P2Y1-specific antagonist MRS2179. Cyst growth was reduced by ∼50% when ATP was removed from the culture medium with apyrase, although stable analogs of ATP failed to increase cyst size. The nonselective P2X receptor inhibitor Coomassie brilliant blue G was ineffective at reducing cyst growth, suggesting no involvement of P2X receptors. Finally, the presence of selective inhibitors of ERK activation (either PD98059 or U0126) greatly reduced cyst growth, whereas in untreated cysts ERK activity was observed to increase with time. We conclude that stimulation of endogenous P2Y receptors by extracellular ATP increases growth of MDCK cysts via cAMP-dependent activation of the ERK pathway. P2Y receptor antagonists may have therapeutic potential in reducing cyst size and slowing disease progression; although further studies in vitro and in vivo are needed to investigate the specificity and role of these P2Y receptors in renal cystic diseases.

The molecular identification and characterization of the adenosine triphosphate (ATP)-sensitive family of P2 receptors is comparatively new. There are two main subgroups, each with several subtypes and widespread tissue distribution, including the kidney. A unique member of the P2X subgroup of P2 receptors is the ATP-gated ion channel P2X(7), which on activation can cause cell blebbing, cytokine release, and cell death by necrosis or apoptosis. We report expression of this receptor in normal rat kidney and in two chronic models of glomerular injury: streptozotocin-induced (STZ) diabetes and ren-2 transgenic (TGR) hypertension.At different time points in these models, we used a polyclonal antibody to the P2X(7) receptor and immunohistochemistry to determine its expression and distribution. We also used Western blotting and real-time polymerase chain reaction (PCR) to detect changes in P2X(7) receptor protein and mRNA expression, respectively.We found only low-level glomerular immuno-staining for the P2X(7) receptor in normal rat kidney, but intense P2X(7) receptor immunostaining of glomeruli in kidneys from diabetic animals at 6 and 9 weeks, and in hypertensive animals at 12 weeks. In diabetic animals, real-time PCR demonstrated a approximately tenfold increase in glomerular P2X(7) receptor mRNA relative to control, and Western blotting confirmed an increase in protein. Immunohistochemistry and immunoelectron microscopy showed staining of glomerular podocytes, which was both intracellular and at the plasma membrane.We conclude that the P2X(7) receptor is not expressed appreciably under normal conditions, but that following glomerular injury it is significantly up-regulated, mainly in podocytes, though also in endothelial and mesangial cells, of animals with STZ-induced diabetes mellitus or TGR hypertension. Although the exact function and regulation of this receptor remain unclear, its association with inflammatory cytokine release and cell death suggests that increased expression might be involved in the pathogenesis of glomerular cell injury or repair.