JTZ-951 (enarodustat), a hypoxia-inducible factor prolyl hydroxylase inhibitor, improves iron utilization and anemia of inflammation: Comparative study against recombinant erythropoietin in rat.

Anemia with inflammation-induced defective iron utilization is a pathological condition observed in patients suffering from chronic kidney disease (CKD) or chronic inflammatory disease. There is no reasonable treatment for these conditions, because the effects of erythropoiesis stimulating agents (ESAs) or iron supplementation in the treatment of anemia are insufficient. JTZ-951 (enarodustat) has been characterized as a novel, orally bioavailable inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH), and has been developed as a novel therapeutic agent for anemia with CKD. In this study, the effects of JTZ-951 on iron utilization during erythropoiesis and on anemia of inflammation were compared with those of recombinant human erythropoietin (rHuEPO) using normal rat and rat model of anemia of inflammation. In normal rats, under conditions in which JTZ-951 and rHuEPO showed similar erythropoietic effect, repeated doses of JTZ-951 induced erythropoiesis while retaining the hemoglobin content in red blood cells, while administration of rHuEPO resulted in decrease in some erythrocyte-related parameters. As for iron-related parameters during erythropoiesis, JTZ-951 exhibited more efficient iron utilization compared to rHuEPO. A single dose of JTZ-951 resulted in decrease in hepcidin expression observed within 24 h after administration, but a single dose of rHuEPO did not. In a rat model of anemia of inflammation (also known as a model with functional iron-deficiency), JTZ-951 showed erythropoietic effect, in contrast with rHuEPO. These results suggest that, unlike rHuEPO, JTZ-951 stimulates erythropoiesis by increasing iron utilization, and improves anemia of inflammation.

Oxidative stress associated with altered activity of glutathione peroxidase and superoxide dismutase enzymes with IDA during pregnancy.

Iron deficiency anemia (IDA) during pregnancy, although associated with disturbances of hematological parameters, is now also considered as a source of oxidative stress (OS). Present study aims to detect any alteration in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes activity in pregnant women with IDA. Levels of GSH-Px and SOD were measured in 156 anemic, pregnant women and compared with similar levels in 20 non anemic, pregnant women. Activity of SOD was found to be reduced in the anemic group when compared with the control group. We found a non- significant increase in GSH-Px activities in the anemic group. These findings could be explained in terms of OS under hypoxic condition which preserves the activity of GSH-Px with a decrease activity of SOD. A positive association was seen between IDA during pregnancy and OS with results suggesting that, apart from the deficiency of iron, some other factors are also associated for the increased OS seen during pregnancy.

Cystathionine ß-synthase is required for body iron homeostasis.

Cystathionine ß-synthase (CBS) catalyzes the transsulfuration pathway and contributes, among other functions, to the generation of hydrogen sulfide. In view of the exceptionally high expression of CBS in the liver and the common interleukin-6 pathway used in the regulatory systems of hydrogen sulfide and hepcidin, we speculate that CBS is involved in body iron homeostasis. We found that CBS knockout (CBS-/- ) mice exhibited anemia and a significant increase in iron content in the serum, liver, spleen, and heart, along with severe damage to the liver, displaying a hemochromatosis-like phenotype. A high level of hepatic and serum hepcidin was also found. A major cause of the systemic iron overload is the reduced iron usage due to suppressed erythropoiesis, which is consistent with an increase in interleukin-6 and reduced expression of erythropoietin. Importantly, in the liver, absence of CBS caused both a reduction in the transcriptional factor nuclear factor erythroid 2-related factor-2 and an up-regulation of hepcidin that led to a decrease in the iron export protein ferroportin 1. The resulting suppression of iron export exacerbates iron retention, causing damage to hepatocytes. Finally, administration of CBS-overexpressing adenovirus into CBS mutant mice could partially reverse the iron-related phenotype. CONCLUSION: Our findings point to a critical role of CBS in iron homeostasis of the body, and the liver in particular; it is likely that a hemochromatosis-like phenotype in patients can be induced by aberration not only in the expression of key molecules in the hepcidin pathway but also of those related to CBS. (Hepatology 2018;67:21-35).

Fetal iron deficiency and genotype influence emotionality in infant rhesus monkeys.

BACKGROUND: Anemia during the third trimester of fetal development affects one-third of the pregnancies in the United States and has been associated with postnatal behavioral outcomes. This study examines how fetal iron deficiency (ID) interacts with the fetal monoamine oxidase A (MAOA) genotype. MAOA metabolizes monoamine neurotransmitters. MAOA polymorphisms in humans affect temperament and modify the influence of early adverse environments on later behavior. OBJECTIVE: The aim of the study was to advance translation of developmental ID research in animal models by taking into account genetic factors that influence outcomes in human populations. METHODS: Male infant rhesus monkeys 3-4 mo old born to mothers fed an ID (10 ppm iron) diet were compared with controls (100 ppm iron). Infant monkeys with high- or low-transcription rate MAOA polymorphisms were equally distributed between diet groups. Behavioral responses to a series of structured experiences were recorded during a 25-h separation of the infants from their mothers. RESULTS: Infant monkeys with low-transcription MAOA polymorphisms more clearly demonstrated the following ID effects suggested in earlier studies: a 4% smaller head circumference, a 39% lower cortisol response to social separation, a 129% longer engagement with novel visual stimuli, and 33% lesser withdrawal in response to a human intruder. The high MAOA genotype ID monkeys demonstrated other ID effects: less withdrawal and emotionality after social separation and lower "fearful" ratings. CONCLUSION: MAOA × ID interactions support the role of monoamine neurotransmitters in prenatal ID effects in rhesus monkeys and the potential involvement of common human polymorphisms in determining the pattern of neurobehavioral effects produced by inadequate prenatal nutrition.

Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia.

Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.

Antioxidant enzymes and oxidative stress in the erythrocytes of iron deficiency anemic patients supplemented with vitamins.

BACKGROUND: Iron deficiency anemia is one of the major causes of morbidity and mortality worldwide. Evidences from epidemiological and clinical studies suggest a possible correlation between antioxidant levels and the anemic disease risk. The present work is to investigate antioxidant levels and lipid peroxidation in anemic patients. METHODS: A number of 30 patients (15 males and 15 females) were selected for the study. Likewise, 30 age- and gender-matched healthy volunteers (15 males and 15 females) were selected with their informed consent. Patients and healthy subjects were supplemented with vitamins C and E for 15 days. The lipid peroxidation both in plasma and erythrocyte lysates was determined by thiobarbituric acid reactive substances and lipid peroxides. The antioxidant vitamins A, C, and E and total antioxidant activity were also analyzed. The antioxidant enzyme superoxide dismutase, catalase, and glutathione peroxidase were also determined. RESULTS: Based on analysis, we found that the increase in lipid peroxidation was higher in the anemic subjects before vitamin supplementation, which was statistically significant at P<0.05. The antioxidant enzymes were higher in the patients before antioxidant supplementation when compared with patients after vitamin supplementation. CONCLUSION: Our data revealed higher oxidative stress before vitamin supplementation in iron deficiency anemic patients and after supplementation, lower lipid peroxidation and increased antioxidant vitamins were achieved.

Inactive matriptase-2 mutants found in IRIDA patients still repress hepcidin in a transfection assay despite having lost their serine protease activity.

Mutations of the TMPRSS6 gene, which encodes Matriptase-2, are responsible for iron-refractory iron-deficiency anemia. Matriptase-2 is a transmembrane protease that downregulates hepcidin expression. We report one frameshift (p.Ala605ProfsX8) and four novel missense mutations (p.Glu114Lys, p.Leu235Pro, p.Tyr418Cys, p.Pro765Ala) found in IRIDA patients. These mutations lead to changes in both the catalytic and noncatalytic domains of Matriptase-2. Analyses of the mutant proteins revealed a reduction of autoactivating cleavage and the loss of N-Boc-Gln-Ala-Arg-p-nitroanilide hydrolysis. This resulted either from a direct modification of the active site or from the lack of the autocatalytic cleavage that transforms the zymogen into an active protease. In a previously described transfection assay measuring the ability of Matriptase-2 to repress the hepcidin gene (HAMP) promoter, all mutants retained some, if not all, of their transcriptional repression activity. This suggests that caution is called for in interpreting the repression assay in assessing the functional relevance of Matriptase-2 substitutions. We propose that Matriptase-2 activity should be measured directly in the cell medium of transfected cells using the chromogenic substrate. This simple test can be used to determine whether a sequence variation leading to an amino acid substitution is functionally relevant or not.

Paraoxonase and arylesterase activities in children with iron deficiency anemia and vitamin B12 deficiency anemia.

Paraoxonase-1 is an esterase enzyme and it has 3 types of activity, namely paraoxonase, arylesterase, and diazoxonase. It has been reported that paraoxonase-1 deficiency is related to increased susceptibility to development of atherosclerosis and cardiovascular disease. The aim of this study was to investigate serum paraoxonase and arylesterase activities in children with iron deficiency anemia and vitamin B(12) deficiency anemia. Thirty children with iron deficiency anemia, 30 children with vitamin B(12) deficiency anemia, and 40 healthy children aged 6 months to 6 years were enrolled in this study. Serum paraoxonase and arylesterase activities were measured with a spectrophotometer by using commercially available kits. Mean paraoxonase and arylesterase activities in vitamin B(12) deficiency anemia group (103 ± 73 and 102 ± 41 U/L, respectively) were significantly lower than mean activities of control group (188 ± 100 and 147 ± 34 U/L, respectively; P < .001 for both) and iron deficiency anemia group (165 ± 103 and 138 ± 39 U/L, respectively; P < .05, P < .001), whereas there were no significant differences between iron deficiency anemia and control groups (P > .05). Paraoxonase and arylesterase activities significantly increased after treatment with vitamin B(12) in vitamin B(12) deficiency anemia; however, there were no significant changes in the activities of these enzymes after iron treatment in iron deficiency anemia group. Important correlations were found between vitamin B(12) levels and both paraoxonase and arylesterase activities (r = .367, P < .001; r = .445, P < .001). Our results suggest that vitamin B(12) deficiency anemia causes important reductions in paraoxonase and arylesterase activities, and after vitamin B(12) therapy the activities of these enzymes returned to near-normal levels.

Correction of iron deficiency anemia in pregnancy and its effects on superoxide dismutase.

Iron deficiency anemia (IDA) affects not only the hematological parameters but also disturb the oxidative balance of body. In pregnancy, this is much more considerable as oxidative stress is considered to be one of the physiological changes during this period. This study aims to observe the effect of daily iron supplement on oxidative stress in pregnancy. In this study, 30 pregnant women with IDA were treated with daily oral iron supplements for 12 weeks. After 12 weeks hemoglobin (Hb), hematocrit, serum ferritin concentration (SFC) and superoxide dismutase (SOD) activity were measured and compared with pre-supplement levels. A significant increase in all the parameters was observed after treatment (p<0.05). When post supplemental values of SFC and SOD were compared with control group comprising of 10 non anemic pregnant women, they were significantly low (p<0.05). Our results indicate that oxidative stress in pregnancy associated with IDA decreases with daily iron supplements but fail to reach normal pregnant levels. This supports iron over load theory in daily iron supplements and suggests that oxidative stress increases if pregnancy is associated with iron deficiency.

Influence of prenatal iron deficiency and MAOA genotype on response to social challenge in rhesus monkey infants.

Social and emotional behaviors are known to be sensitive to both developmental iron deficiency (ID) and monoamine oxidase A (MAOA) gene polymorphisms. In this study, male rhesus monkey infants deprived of dietary iron in utero were compared with iron sufficient (IS) controls (n = 10/group). Half of each group had low MAOA activity genotypes and half had high MAOA activity genotypes. A series of social response tests were conducted at 3-14 months of age. MAOA genotype influenced attention to a video of aggressive behavior, emotional expression (fear, grimace and sniff) in the social intruder test, social actions (displacement, grooming) in the social dyad test, and aggressive responses to a threatening picture. Interactions between MAOA and prenatal ID were seen in response to the aggressive video, in temperament ratings, in affiliative behavior in the social dyad test, in cortisol response in the social buffering test and in response to a social intruder and to pictures with social and nonsocial themes. In general, the effects of ID were dependent on MAOA genotype in terms of both direction and size of the effect. Nutrition/genotype interactions may shed new light on behavioral consequences of nutritional deprivation during brain development.

A novel mutation Gly603Arg of TMPRSS6 in a Korean female with iron-refractory iron deficiency anemia.

Iron-refractory iron deficiency anemia (IRIDA) is a rare hereditary form of IDA with autosomal recessive inheritance. IRIDA is characterized by hypochromic microcytic anemia unresponsive to oral iron treatment, low transferrin saturation, and a high level of iron-regulated hormone hepcidin. The genetic background of IRIDA is mutations in the TMPRSS6 gene encoding matriptase-2 (TMPRSS6) that prevent inactivation of hemojuvelin, an activator of hepcidin transcription. We herein report a Korean female with IRIDA who was compound heterozygous for two mutations in TMPRSS6: a novel missense mutation c.1807G>C (p.Gly603Arg) in the serine protease domain and a known splicing mutation c.863+1G>T (IVS6+1G>T).

Se bioavailability and glutathione peroxidase activity in iron deficient rats.

Little information is available on the relationship of Se deposit in target organs and GPx activity in iron deficiency anemia. As red blood cells (RBCs) play a crucial role on Se metabolism and during Fe deficiency anemia a lower count of RBCs is featured, we aimed to investigate the influence of this pathology on Se bioavailability and the relationship with antioxidant status. 20 male Wistar rats were randomly divided into two groups, a control group receiving AIN-93G diet with normal Fe content (45mg/kg diet) and the Fe-deficient group receiving AIN-93G diet with low Fe content (5mg/kg diet) for 40 days. Both diets were prepared with an adequate Se content (0.180mg/kg diet). The digestive and metabolic utilization of Se, the distribution in target organ, the GPx activity and TBARS production were measured after receiving the diets. Se retention increased (P<0.001) in the anemic group, fact that contributes to keep the enzymatic antioxidant activity of GPx in normal levels and the tendency observed is that stored Se increased in the organs, especially in kidney (P<0.01), however, a lower Se deposit was found in sternum of anemic rats (P<0.001). The lower count of RBCs featured in this pathology (P<0.001) causes a decrease of Se concentration in sternum meanwhile the increase in kidney deposit is a consequence of the lower urinary losses (P<0.001).

Regulation of type II transmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors: new link between hypoxia signaling and iron homeostasis.

Hepcidin is a liver-derived hormone with a key role in iron homeostasis. In addition to iron, it is regulated by inflammation and hypoxia, although mechanisms of hypoxic regulation remain unclear. In hepatocytes, hepcidin is induced by bone morphogenetic proteins (BMPs) through a receptor complex requiring hemojuvelin (HJV) as a co-receptor. Type II transmembrane serine proteinase (TMPRSS6) antagonizes hepcidin induction by BMPs by cleaving HJV from the cell membrane. Inactivating mutations in TMPRSS6 lead to elevated hepcidin levels and consequent iron deficiency anemia. Here we demonstrate that TMPRSS6 is up-regulated in hepatic cell lines by hypoxia and by other activators of hypoxia-inducible factor (HIF). We show that TMPRSS6 expression is regulated by both HIF-1α and HIF-2α. This HIF-dependent up-regulation of TMPRSS6 increases membrane HJV shedding and decreases hepcidin promoter responsiveness to BMP signaling in hepatocytes. Our results reveal a potential role for TMPRSS6 in hepcidin regulation by hypoxia and provide a new molecular link between oxygen sensing and iron homeostasis.

A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA.

IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated.

[Iron deficiency anaemia due to a matriptase-2 mutation]. / IJzergebreksanemie door een matriptase 2-mutatie.

A 36-year old female patient who had had iron deficiency anaemia since her childhood showed no clear response to oral iron treatment. Elevated serum hepcidin levels were found after excluding other causes of iron deficiency. This is in contrast to what is expected in iron deficiency anaemia and indicates a primary defect in hepcidin regulation. Indeed, in the search for a defect in genes coding for hepcidin-regulating proteins the patient was found to be compound heterozygous for two different mutations in the TMPRSS6 gene. This leads to a dysfunctional matriptase-2 protein for which the gene codes. Consequently, liver cells cannot inhibit hepcidin production in the presence of low serum iron levels. High hepcidin levels result in less iron being absorbed from the bowel than is necessary for erythropoiesis. Therefore, patients with matriptase-2 deficiency respond poorly to oral iron treatment and have to be treated with intravenous iron.

Novel TMPRSS6 mutations associated with iron-refractory iron deficiency anemia (IRIDA).

Mutations leading to abrogation of matriptase-2 proteolytic activity in humans are associated with an iron-refractory iron deficiency anemia (IRIDA) due to elevated hepcidin levels. In this paper we describe 12 IRIDA patients belonging to 7 unrelated families and identify 10 (9 novel) TMPRSS6 mutations spread along the gene sequence: 5 missense, 1 non sense and 4 frameshift. The frameshift and non sense mutations are predict to result in truncated protein lacking the catalytic domain. The causal role of missense mutations (Y141C, I212T, R271Q, S304L and C510S) is demonstrated by in silico analysis, their absence in 100 control chromosomes and the high conservation of the involved residues. The C510S mutation in the LDLRA domain in silico model causes an intra-molecular structural imbalance that impairs matriptase-2 activation. We also assessed the in vitro effect on hepcidin promoter and the proteolytic activity of I212T and R271Q variants demonstrating a reduced inhibitory effect for the former mutation, but surprisingly a normal function for R271Q which appears a silent mutation in vitro. Based on mRNA expression studies I212T could also decrease the total amount of protein produced, likely interfering with mRNA stability. Collectively, our results extend the pattern of TMPRSS6 mutations associated with IRIDA and propose a model of causality for some of the novel missense mutation.

Proatherogenic disturbances in lipoprotein profile, associated enzymes and transfer proteins in women with iron deficiency anaemia.

OBJECTIVE: To characterize the lipid-related atherogenic risk factors in iron deficiency anaemia (IDA) patients. DESIGN AND METHODS: Twenty IDA women were compared to healthy age-matched controls. Lipoprotein profile, cholesteryl ester transfer protein (CETP), paraoxonase (PON) 1 and lipoprotein-associated phospholipase A(2) (LpPLA(2)) activities and plasma levels of oxidized-LDL were evaluated. RESULTS: Triglycerides were higher (median [range]) (1.0 [0.5-1.9] vs. 0.7 [0.5-1.5] mmol/L, p<0.05) and HDL-C lower (mean + or - SD) (1.3 + or - 0.3 vs. 1.6 + or - 0.4 mmol/L, p<0.01) in the patients group. CETP (197 + or - 29% vs. 151 + or - 29% mL(-1) h(-1), p<0.001), PON 1 (122 + or - 17 vs. 140 + or - 33 micromol mL(-1) min(-1), p<0.05) and LpPLA(2) (9.6 + or - 2.0 vs. 8.1 + or - 1.7 micromol mL(-1) h(-1), p<0.05) activities were different in IDA women. No difference was observed in oxidized-LDL. Haemoglobin correlated negatively with triglycerides (r=-0.35, p<0.05), CETP (r=-0.62, p<0.001) and LpPLA(2) (r=-0.34, p<0.05), while ferritin was positively associated with HDL-C (r=0.39, p<0.05) and inversely with CETP (r=-0.49, p<0.005). CONCLUSION: The alterations in lipoprotein profile, CETP, PON 1 and LpPLA(2) activities described in the present study indicate that non-treated IDA might represent a proatherogenic state.

Role of matriptase-2 (TMPRSS6) in iron metabolism.

Iron, an essential element for life, is regulated primarily at the level of uptake, storage, and transport in order to maintain sufficient availability for normal physiology. The key protein in iron homeostasis is a 25-amino-acid peptide, hepcidin, which modulates the amount of iron in the circulation by binding and promoting the degradation of the iron exporter ferroportin. Given the central importance of hepcidin, recent studies have focused on how iron is sensed and how the iron signal is transmitted to hepcidin. Mutations in a type II serine protease, matriptase-2/TMPRSS6, were recently identified to be associated with severe iron deficiency caused by inappropriately high levels of hepcidin expression. A key biologically relevant substrate for the proteolytic activity of matriptase-2/TMPRSS6 was found to be hemojuvelin, a cell surface protein that regulates hepcidin expression through a BMP/SMAD pathway. In this review, we discuss the putative role of matriptase-2/TMPRSS6 in iron homeostasis.