Iron-restricted Diet Research Articles

Abstract TMP114: The Role of Iron Mediated Oxidative Stress and Lipid Peroxidation in the Development of Intracranial Aneurysm Rupture

Introduction: Iron is an essential element for vascular wall maintenance. However, iron deposition in the vascular wall may cause oxidative stress, which can attack DNA, proteins, and lipid membranes via the Fenton/Haber-Weiss reaction. Iron-induced oxidative stress triggers lipid oxidation, inflammation, endothelial cell activation and intracranial aneurysmal formation, growth, and rupture. Subarachnoid hemorrhage from intracranial aneurysm rupture results in significant morbidity and mortality. This study used a mouse model of intracranial aneurysm to evaluate the effect of dietary iron restriction on aneurysm formation and rupture. Methods: Intracranial aneurysms were induced using deoxycorticosterone acetate-salt-induced hypertension and a single injection of elastase into the cerebrospinal fluid of the basal cistern. Mice were fed an iron-restricted diet or a normal diet. Aneurysm rupture was detected by neurological symptoms, while the presence of intracranial aneurysm with subarachnoid hemorrhage was confirmed by post-mortem examination. Results: The aneurysmal rupture rate was significantly lower in iron-restricted diet mice (37%) compared with normal diet mice (76%; p < 0.05). Serum oxidative stress, iron accumulation, macrophage infiltration, and 8-hydroxy-2′-deoxyguanosine in the vascular wall were lower in iron-restricted diet mice ( p < 0.01). The areas of iron positivity were similar to the areas of CD68 positivity and 8-hydroxy-2′-deoxyguanosine in both normal diet and iron-restricted diet mice aneurysms. Conclusions: Compared with a normal diet, an iron-restricted diet reduced the incidence of intracranial aneurysmal rupture in mice via inhibition of iron accumulation in the aneurysmal wall. Iron may be a potential therapeutic target to prevent intracranial aneurysm rupture. Long-term iron restriction may be required to affect aneurysm formationrupture.

Dietary Iron Restriction Protects against Aneurysm Rupture in a Mouse Model of Intracranial Aneurysm

Introduction: Iron accumulation in vessel walls induces oxidative stress and inflammation, which can cause cerebrovascular damage, vascular wall degeneration, and intracranial aneurysmal formation, growth, and rupture. Subarachnoid hemorrhage from intracranial aneurysm rupture results in significant morbidity and mortality. This study used a mouse model of intracranial aneurysm to evaluate the effect of dietary iron restriction on aneurysm formation and rupture. Methods: Intracranial aneurysms were induced using deoxycorticosterone acetate-salt-induced hypertension and a single injection of elastase into the cerebrospinal fluid of the basal cistern. Mice were fed an iron-restricted diet (n = 23) or a normal diet (n = 25). Aneurysm rupture was detected by neurological symptoms, while the presence of intracranial aneurysm with subarachnoid hemorrhage was confirmed by post-mortem examination. Results: The aneurysmal rupture rate was significantly lower in iron-restricted diet mice (37%) compared with normal diet mice (76%; p < 0.05). Serum oxidative stress, iron accumulation, macrophage infiltration, and 8-hydroxy-2′-deoxyguanosine in the vascular wall were lower in iron-restricted diet mice (p < 0.01). The areas of iron positivity were similar to the areas of CD68 positivity and 8-hydroxy-2′-deoxyguanosine in both normal diet and iron-restricted diet mouse aneurysms. Conclusions: These findings suggest that iron is involved in intracranial aneurysm rupture via vascular inflammation and oxidative stress. Dietary iron restriction may have a promising role in preventing intracranial aneurysm rupture.

PS-B11-10: IRON DEFICIENCY AFFECTS CARDIAC FUNCTION BUT HAS NO EFFECT ON BLOOD PRESSURE IN MICE WITH METABOLIC SYNDROME

Objective: Metabolic syndrome is a serious problem concerning public health, increasing the risk of cardiovascular diseases. Some epidemiological studies have reported that iron overload is associated with metabolic syndrome; however, it remains unknown whether iron deficiency affects the pathogenesis of metabolic syndrome. Therefore, in this study, we investigated the impact of dietary iron deficiency in genetically obese KKAy mice, a model of human metabolic syndrome. Methods: Four-week-old male KKAy mice and control C57BL/6J mice (wild-type mice, WT) were fed a normal or an iron-restricted diet, and the impact of dietary iron deficiency was investigated 12 weeks after diet initiation. Results: Compared with WT mice, systolic blood pressure presented an increasing trend in KKAy mice; however, the difference was not statistically significant between the two groups, and this trend did not change after iron restriction. Notably, some KKAy mice died following iron restriction, while no deaths were recorded in WT mice. Of interest, obese KKAy mice exhibited cardiac hypertrophy and cardiac dysfunction, thereby developing heart failure with pulmonary congestion after iron restriction. Conclusions: These results indicate that iron deficiency affects cardiac function in metabolic syndrome.

Dietary Iron Restriction Alters Microbiota-Host Crosstalk and Protects Against Vaso-Occlusion and Organ Damage in Sickle Cell Disease Mice

Introduction: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin, and consequently improved hemolysis and ameliorated anemia. Our lab previously demonstrated that microbial signals from the gut also participated in initiation of VOE and organ damage in SCD mice. To elucidate connections between dietary iron, the microbiota-gut axis, and SCD pathogenesis, we hypothesize that dietary iron restriction may reduce microbiota load and gut permeability, and contributes to amelioration of acute and chronic complications in SCD mice. Methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were conducted 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration in SCD compared to Ctrl group(0.6±0.1 v.s. 4.0±0.7ug iron/mg liver tissue; P=0.001), confirming that dietary iron restriction reverses organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (19.7±0.3 v.s. 20.1±0.2 pg; P=0.006) and serum indirect bilirubin (0.4±0.04 v.s. 0.7±0.07 mg/dL; P=0.001) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice which corresponded with improved VOE parameters (RBC velocity: 2.0±0.2 v.s. 1.6±0.1mm/s; P=0.01; blood flow rate: 314±33 v.s. 230±13pL/s; P=0.01; leukocyte adhesion: 10±1 v.s. 14±1cells/100um vein; P=0.01; IRD v.s. Ctrl). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig1). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig2B). We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds. Microbiota load in IRD treated SCD mice were decreased to 25% of Ctrl level. IRD inhibited translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability. We detected significantly decreased TLR2 ligands in serum of IRD compared to Ctrl mice (0.31±0.04 v.s. 0.46±0.05 TLR2 ligand activities O.D. 650nm; P=0.04) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments under development focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related and novel therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dietary iron restriction protects against vaso-occlusion and organ damage in murine sickle cell disease

AbstractSickle cell disease (SCD) is an inherited disorder resulting from a β-globin gene mutation, and SCD patients experience erythrocyte sickling, vaso-occlusive episodes (VOE), and progressive organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron overload, and another subpopulation of SCD patients manifest iron deficiency. To elucidate connections between dietary iron, the microbiome, and SCD pathogenesis, we treated SCD mice with an iron-restricted diet (IRD). IRD treatment reduced iron availability and hemolysis, decreased acute VOE, and ameliorated chronic organ damage in SCD mice. Our results extend previous studies indicating that the gut microbiota regulate disease in SCD mice. IRD alters microbiota load and improves gut integrity, together preventing crosstalk between the gut microbiome and inflammatory factors such as aged neutrophils, dampening VOE, and organ damage. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments, which are under development, focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related- and novel-therapeutic targets.

PI-10: DIETARY IRON RESTRICTION PROTECTS AGAINST VASO-OCCLUSION AND ORGAN DAMAGE IN SICKLE CELL DISEASE

Purpose: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin as reflected by reduced RBC mean corpuscular hemoglobin concentration (MCHC), and consequently improved hemolysis and ameliorated anemia. Understanding iron homeostasis is therefore critical to SCD pathogenesis and holds the promise for identifying novel approaches to treat SCD. Materials and methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were done 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration (Fig-1A) in SCD compared to Ctrl group, confirming that dietary iron restriction ameliorates organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (Fig-1B) and serum indirect bilirubin(IDBILC) (Fig-1C) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice (Fig-1D), which corresponded with improved VOE parameters. Specifically, intravital microscopy (IVM) demonstrated elevated mean centerline RBC velocity and blood flow rate and decreased leukocyte adhesion in IRD compared to Ctrl mice (Fig-1E-G). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig-1H). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig-2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig-2B,C), decreased serum liver enzymes (ALT, AST; Fig-2D), and reduced levels of total and direct bilirubin (TBILC, DBILC; Fig-2D).We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds.IRD inhibits translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability (Fig-2E). We detected significantly decreased TLR2 ligands in serum of IRD mice (Fig-2F) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates for the first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. Novel approaches that target iron availability/absorption in the gut, the gut microbiome and aged neutrophil crosstalk and gut barrier integrity, provide an important area of further investigation with the potential of improving hemolysis, VOE, and organ damage in SCD patients.(A)The bone-marrow/spleen/liver Non-heme iron content. (B)MCHC, (C)IDBILC,(D)aged neutrophils percentage in Ctrl/IRD SCD mice. IVM parameters (E) (Vrbc) (F) blood flow rate (J) the number of adherent leukocytes and (J) survival rate after TNF-a injection(A)Leukocyte infiltration in lung and (B)liver tissue. (C)Liver necrosis, leukocyte infiltration, and fibrosis. (D)Liver ALT and AST, TBILC, and DBILC from sera. (E)serum FITC-Dextran and (F) TLR2 ligands activity in Ctrl/IRD SCD mice The authors do not declare any conflict of interest

Abstract TP256: The Role Of Dietary Iron Restriction In The Pathophysiology Of Intracranial Aneurysm Rupture

Background: Iron is an essential element for maintaining vascular wall, however iron depositions in vascular wall cause tissue damage by oxidative stress via Fenton/Haber-Weiss reaction. We hypothesized that iron promotes intracranial aneurysmal formation and rupture through oxidative stress and vascular inflammation. Using a mouse model of intracranial aneurysm, we examined whether iron restriction reduces aneurysm formation and rupture. Methods: To induce aneurysm, we combined induced systemic hypertension (deoxycorticosterone acetate-salt hypertension) and a single injection of elastase (35mU) into the cerebrospinal fluid at the right basal cistern. The mice were divided into two groups and fed with iron restricted diet (IRD) (0.32mg/100g) or normal diet (ND) (600mg/100g) for 5 weeks. Results: IRD significantly reduced the aneurysmal rupture compared to ND (ND versus IRD: 76% versus 37%; P &lt; 0.05). Whereas, there was no significant difference in the overall incidence of aneurysms between the ND and IRD groups (ND versus IRD: 68% versus 69%; P =1.00). Conclusion: Our data demonstrate that iron restriction reduces intracranial aneurysm rupture.

Effects of dietary iron restriction on kidney mitochondria function and oxidative stress in streptozotocin-diabetic rats

Diabetes mellitus is characterized by chronic hyperglycemia causing mitochondrial dysfunction and kidney iron overload has been observed during diabetes. We evaluated the effects of an iron-restricted diet (IRD) on mitochondrial function, oxidative stress, and mitochondrial iron levels in the kidneys of Wistar rats with streptozotocin-induced diabetes. IRD ameliorated mitochondrial dysfunction in diabetic rats by restoring mitochondrial respiration and respiratory complex activity, improving oxidative stress and glutathione status in kidney mitochondria. We also observed mitochondrial iron overload. Our data suggest that elevated iron levels were attenuated by IRD, resulting in modulation of oxidative stress and mitochondrial function in the kidney.

Iron-deficient diet induces distinct protein profile related to energy metabolism in the striatum and hippocampus of adult rats

ABSTRACT Iron deficiency is a public health problem that affects all age groups. Its main consequence is anemia, but it can also affect cognitive functions. Although the negative effects of iron deficiency on cognitive function have been extensively described, the underlying mechanism has not been fully investigated. Thus, to gain an unbiased insight into the effects of iron deficiency (ID) on discrete brain regions, we performed a proteomic analysis of the striatum and hippocampus of adult rats subjected to an iron restricted (IR) diets for 30 days. We found that an IR diet caused major alterations in proteins related to glycolysis and lipid catabolism in the striatum. In the hippocampus, a larger portion of proteins related to oxidative phosphorylation and neurodegenerative diseases were altered. These alterations in the striatum and hippocampus occurred without a reduction in local iron levels, although there was a drastic reduction in liver iron and ferritin. Moreover, the IR group showed higher fasting glycaemia than the control group. These results suggest that brain iron content is preserved during acute iron deficiency, but the alterations of other systemic metabolites such as glucose may trigger distinct metabolic adaptations in each brain region. Abnormal energy metabolism precedes and persists in many neurological disorders. Thus, altered energy metabolism can be one of the mechanisms by which iron deficiency affects cognitive functions.

Iron Deficiency Induces Faster Sickling Kinetics in Transgenic Sickle Mice

Sickle cell disease (SCD) is caused by polymerization of deoxygenated hemoglobin‐S (HbS) within the red blood cell (RBC). The kinetics of hemoglobin polymerization are dependent on the intra‐erythrocytic concentration of HbS; specifically, the time from deoxygenation to polymerization is inversely related to HbS concentration30. We hypothesized that iron‐deficient erythropoiesis would produce RBCs with decreased intracellular Hb concentration and prolonged sickling times which would reduce the severity of SCD.To interrogate this hypothesis, we studied Townes mice that express humanized alpha globin, gamma globin, and either beta‐S (Townes‐SCD) or normal beta globin (Townes‐Control). RBCs from these Townes‐SCD mice have similar sickling kinetics to RBCs from human volunteers with SCD. To induce iron deficiency, we fed mice either an iron‐restricted diet (3 ppm iron) or iron‐normal diet (48 ppm iron) introduced at the time of weaning. After two months on the experimental diets, we measured RBC parameters using an Advia 2120 hematology analyzer and measured in vitro RBC sickling kinetics at 3% oxygen. Analysis of RBC parameters revealed that SCD mice on the iron‐restricted diet had lower hematocrit (32% vs 42.2%, P &lt;0.0001), and there were trends toward lower hemoglobin, mean corpuscular volume, mean cell hemoglobin, and mean corpuscular hemoglobin concentration. Surprisingly, the in vitro sickling kinetic assay showed a significantly shortened time to 50% sickled in the Iron Restricted group (t50: 96.6 vs 136.5 min., p&lt;0.0001).At this two‐month time point, this study provides insights into the hematologic changes that occur during the initial onset of iron deficiency anemia in Townes‐SCD mice. We were surprised to find that the rate of sickling increased in the iron‐restricted mice; however, at two months of iron deficient diet, we have yet to see a substantial fall in the MCHC, a major determinant of the sickling delay time. Furthermore, these mice may have experienced compensatory changes in the red cell that adversely affected sickling such as a decrease in hemoglobin F levels, or a shift in the Hb‐ O2 dissociation curve that favors releasing oxygen at lower partial pressures. To address these questions, we will investigate the hemoglobin composition and the oxygen dissociation curve at the remaining four‐ and six‐month timepoints. We aim to determine the intermediate and long‐term potential of iron deficiency to delay sickling and reduce the severity of organ damage in the context of SCD. This may inform a strategy of optimizing iron intake as a potential treatment for SCD.Support or Funding InformationSupport from NIAID Intramural funding and NIH Division of Veterinary ResourcesScholarship Award from the Saudi Arabian Cultural Mission (SACM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deficiency of the BMP Type I receptor ALK3 partly protects mice from anemia of inflammation

BackgroundInflammatory stimuli induce the hepatic iron regulatory hormone hepcidin, which contributes to anaemia of inflammation (AI). Hepcidin expression is regulated by the bone morphogenetic protein (BMP) and the interleukin-6 (IL-6) signalling pathways. Prior results indicate that the BMP type I receptor ALK3 is mainly involved in the acute inflammatory hepcidin induction four and 72 h after IL-6 administration. In this study, the role of ALK3 in a chronic model of inflammation was investigated. The intact, heat-killed bacterium Brucella abortus (BA) was used to analyse its effect on the development of inflammation and hypoferremia in mice with hepatocyte-specific Alk3-deficiency (Alk3fl/fl; Alb-Cre) compared to control (Alk3fl/fl) mice.ResultsAn iron restricted diet prevented development of the iron overload phenotype in mice with hepatocyte-specific Alk3 deficiency. Regular diet leads to iron overload and increased haemoglobin levels in these mice, which protects from the development of AI per se. Fourteen days after BA injection Alk3fl/fl; Alb-Cre mice presented milder anaemia (Hb 16.7 g/dl to 11.6 g/dl) compared to Alk3fl/fl control mice (Hb 14.9 g/dl to 8.6 g/dl). BA injection led to an intact inflammatory response in all groups of mice. In Alk3fl/fl; Alb-Cre mice, SMAD1/5/8 phosphorylation was reduced after BA as well as after infection with Staphylococcus aureus. The reduction of the SMAD1/5/8 signalling pathway due to hepatocyte-specific Alk3 deficiency partly suppressed the induction of STAT3 signalling.ConclusionThe results reveal in vivo, that 1) hepatocyte-specific Alk3 deficiency partly protects from AI, 2) the development of hypoferremia is partly dependent on ALK3, and 3) the ALK3/BMP/hepcidin axis may serve as a possible therapeutic target to attenuate AI.

Influence of dietary iron intake restriction on the development of hypertension in weanling prehypertensive rats.

Hypertension is a major public health problem leading to death. To reduce the morbidity and mortality in patients with hypertension, it is crucial to develop a novel strategy for prevention of hypertension. We have currently reported an attempt at dietary iron intake restriction as non-pharmacological treatment of hypertension in patients with hypertension. However, it remains fully unknown whether dietary iron restriction prevents the development of hypertension. We investigated the influence of dietary iron restriction on the development of hypertension in weanling pre-hypertensive model rats. 3-week-old male stroke-prone spontaneously hypertensive rats (SHR-SP) were randomly divided into two groups and were given an ad libitum normal diet or an iron-restricted diet for 12weeks. Blood pressure was progressively increased in SHR-SP according to growth, while dietary iron restriction attenuated the development of hypertension. Proteinuria was also increased in SHR-SP according to growth, whereas dietary iron restriction suppressed the increment of proteinuria. SHR-SP exhibited glomerulosclerosis and exacerbated renal interstitial fibrosis at 15weeks old, indicating that SHR-SP developed hypertensive nephropathy in the adult stage; however, these changes were attenuated by dietary iron restriction. Gelatin zymography showed dietary iron restriction decreased both renal MMP-2 and MMP-9 activities in SHR-SP at 15weeks old. Of interest, dietary iron restriction suppressed renal TGFβ-RI expression and Smad2 phosphorylation in SHR-SP. Furthermore, dietary iron restriction decreased renal fibrosis, renal MMP-2 and MMP-9 activities, renal TGFβ-RI expression, and Smad2 phosphorylation in rats with unilateral ureteral obstruction. Dietary iron restriction prevented the development of hypertension in weanling pre-hypertensive rats.

Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

Prenatal iron deficiency alters fetal developmental trajectories, which results in persistent changes in organ function. Here, we studied the effects of prenatal iron deficiency on fetal kidney and liver mitochondrial function. Pregnant Sprague-Dawley rats were fed partially or fully iron-restricted diets to induce a state of moderate or severe iron deficiency alongside iron-replete control rats. We assessed mitochondrial function via high-resolution respirometry and reactive oxygen species generation via fluorescence microscopy on gestational d 21. Hemoglobin levels were reduced in dams in the moderate (-31%) and severe groups (-54%) compared with controls, which was accompanied by 55% reductions in fetal hemoglobin levels in both moderate and severe groups versus controls. Male iron-deficient kidneys exhibited globally reduced mitochondrial content and respiration, as well as increased cytosolic superoxide and decreased NO. Female iron-deficient kidneys exhibited complex II down-regulation and increased mitochondrial oxidative stress. Male iron-deficient livers exhibited reduced complex IV respiration and increased cytosolic superoxide, whereas female liver tissues exhibited no alteration in oxidant levels or mitochondrial function. These findings indicate that prenatal iron deficiency causes changes in mitochondrial content and function as well as oxidant status in a sex- and organ-dependent manner, which may be an important mechanism that underlies the programming of cardiovascular disease.-Woodman, A. G., Mah, R., Keddie, D., Noble, R. M. N., Panahi, S., Gragasin, F. S., Lemieux, H., Bourque, S. L. Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

Dietary iron restriction alleviates renal tubulointerstitial injury induced by protein overload in mice

Increased proteinuria causes tubulointerstitial injury due to inflammation in chronic kidney disease (CKD). Iron restriction exhibits protective effects against renal dysfunction; however, its effects against protein overload-induced tubulointerstitial damage remain unclear. Here, we investigated dietary iron restriction effect on tubulointerstitial damage in mice with protein-overload tubulointerstitial injury. Renal tubulointerstitial injury in animal model was induced by intraperitoneal injection of an overdose of bovine serum albumin (BSA). We divided mice into three groups: normal saline + normal diet (ND), BSA + ND, and BSA + iron-restricted diet (IRD). BSA overload induced renal tubulointerstitial injury in the ND mice, which was ameliorated in the IRD mice. Inflammatory cytokines and extracellular matrix mRNA expression was upregulated in BSA + ND mice kidneys and was inhibited by IRD. BSA-induced increase in renal superoxide production, NADPH oxidase activity, and p22phox expression was diminished in the IRD mice. IRD suppression increased BSA-induced renal macrophage infiltration. Moreover, BSA mice exhibited nucleotide-binding oligomerisation domain-like receptor pyrin domain-containing protein (NLRP) inflammasome activation, which was inhibited by IRD. Ferrous iron increased in kidneys with BSA overload and was inhibited by IRD. Thus, iron restriction inhibited oxidative stress and inflammatory changes, contributing to the protective effect against BSA overload-induced tubulointerstitial injury.

Iron-Restricted Diet Affects Brain Ferritin Levels, Dopamine Metabolism and Cellular Prion Protein in a Region-Specific Manner.

Iron is an essential micronutrient for several physiological functions, including the regulation of dopaminergic neurotransmission. On the other hand, both iron, and dopamine can affect the folding and aggregation of proteins related with neurodegenerative diseases, such as cellular prion protein (PrPC) and α-synuclein, suggesting that deregulation of iron homeostasis and the consequential disturbance of dopamine metabolism can be a risk factor for conformational diseases. These proteins, in turn, are known to participate in the regulation of iron and dopamine metabolism. In this study, we evaluated the effects of dietary iron restriction on brain ferritin levels, dopamine metabolism, and the expression levels of PrPC and α-synuclein. To achieve this goal, C57BL/6 mice were fed with iron restricted diet (IR) or with normal diet (CTL) for 1 month. IR reduced iron and ferritin levels in liver. Ferritin reduction was also observed in the hippocampus. However, in the striatum of IR group, ferritin level was increased, suggesting that under iron-deficient condition, each brain area might acquire distinct capacity to store iron. Increased lipid peroxidation was observed only in hippocampus of IR group, where ferritin level was reduced. IR also generated discrete results regarding dopamine metabolism of distinct brain regions: in striatum, the level of dopamine metabolites (DOPAC and HVA) was reduced; in prefrontal cortex, only HVA was increased along with the enhanced MAO-A activity; in hippocampus, no alterations were observed. PrPC levels were increased only in the striatum of IR group, where ferritin level was also increased. PrPC is known to play roles in iron uptake. Thus, the increase of PrPC in striatum of IR group might be related to the increased ferritin level. α-synuclein was not altered in any regions. Abnormal accumulation of ferritin, increased MAO-A activity or lipid peroxidation are molecular features observed in several neurological disorders. Our findings show that nutritional iron deficiency produces these molecular alterations in a region-specific manner and provide new insight into the variety of molecular pathways that can lead to distinct neurological symptoms upon iron deficiency. Thus, adequate iron supplementation is essential for brain health and prevention of neurological diseases.

Iron-restricted pair-feeding affects renal damage in rats with chronic kidney disease.

BackgroundWe have previously shown that dietary iron restriction prevents the development of renal damage in a rat model of chronic kidney disease (CKD). However, iron deficiency is associated with appetite loss. In addition, calorie restriction is reported to prevent the development of end-stage renal pathology in CKD rats. Thus, the beneficial effect of iron restriction on renal damage may depend on calorie restriction. Here, we investigate the effect of pair-feeding iron restriction on renal damage in a rat model of CKD.MethodsFirst, to determine the amount of food intake, Sprague-Dawley (SD) rats were randomly given an ad libitum normal diet or an iron-restricted diet, and the food intake was measured. Second, CKD was induced by a 5/6 nephrectomy in SD rats, and CKD rats were given either a pair-feeding normal or iron-restricted diet.ResultsFood intake was reduced in the iron-restricted diet group compared to the normal diet group of SD rats for 16 weeks (mean food intake; normal diet group and iron-restricted diet group: 25 and 20 g/day, respectively). Based on the initial experiments, CKD rats received either a pair-feeding normal or iron-restricted diet (20 g/day) for 16 weeks. Importantly, pair-feeding iron restriction prevented the development of proteinuria, glomerulosclerosis, and tubulointerstitial damage in CKD rats. Interestingly, pair-feeding iron restriction attenuated renal expression of nuclear mineralocorticoid receptor in CKD rats.ConclusionsPair-feeding iron restriction affected renal damage in a rat model of CKD.

Repeated phlebotomies decrease body iron storage in adult dogs.

The aim of the present study was to determine changes in body iron storage in adult dogs following phlebotomy. We performed repeated phlebotomies by removing 1% body weight (approximately 10% of the total blood volume) weekly for a total of 12 times using adult beagle dogs without an iron-restricted diet. After treatment, stored iron was decreased, as demonstrated by gradual reductions in serum ferritin levels and hepatic iron contents. Anemia and abnormalities in blood chemistry analysis were not observed; therefore, this method was considered safe and useful for control of stored iron levels in adult dogs.

Systems Genetics Analysis of Iron and Its Regulation in Brain and Periphery.

In this contribution, we demonstrate the utility of the systems genetics-systems biology approach to the study of iron regulation while employing a comprehensive database. We describe our work in iron regulation in the brain and periphery under normal iron and iron-restricted dietary conditions in the BXD family of recombinant inbred mouse strains. Using multiple measures, we showed wide variation among the strains in the effect of being fed an iron-restricted diet for 100 days in every measure from brain and from the periphery. All data were entered into GeneNetwork ( www.genenetwork.org ), a database that contains genotypic, phenotypic, and gene expression data (Rosen et al., Methods Mol Biol 401:287-303, 2007). Using this resource, we were able to ask the following four questions concerning possible candidate genes underlying our measures: (1) what is the range of response for each of the measures? (2) Does the pattern of variability show continuous (additive genetic) or discrete (Mendelian) distribution across strains? (3) Are there genetic markers that are associated with the variability in the measures? (4) Are there genes in near the markers that contain associated allelic differences, and whose expression is related to the variability in the measures? Other questions that we could address include: (5) what is the association among the measures between the sexes? (6) What is the association among the measures, e.g., is liver iron status under the diets related to brain iron? (7) What is the relationship between our measures and other phenotypic parameters-i.e., is there an association between our brain iron measures and neurochemical phenotypes extant in the database? And finally, (8) are there gene networks that underlie single or combined measures?

Attenuation of hypertension and renal damage in renovascular hypertensive rats by iron restriction.

Iron is a catalyst in the formation of reactive oxygen species. Oxidative stress is associated with the pathogenesis of both human and experimental animal models of renovascular hypertension. We hypothesized that iron is involved in the pathogenesis of renovascular hypertension and that iron restriction may affect the pathogenesis of renovascular hypertension via the inhibition of oxidative stress. Herein, we investigated the effect of iron restriction on hypertension and renal damage in a rat model of two-kidney one-clip (2K1C) renovascular hypertension. Renovascular hypertension was induced by 2K1C in male Sprague-Dawley rats. At the day of clipping, 2K1C rats were divided into untreated (2K1C) and dietary iron-restricted groups (2K1C+IR). The 2K1C rats showed hypertension after the day of clipping, whereas dietary iron restriction attenuated the development of hypertension. Vascular hypertrophy and the increased fibrotic area were suppressed in the 2K1C+IR group. The clipped kidney developed renal atrophy in both the 2K1C and 2K1C+IR groups after clipping. However, the unclipped kidney showed renal hypertrophy in the 2K1C and 2K1C+IR groups, and the extent was less in the 2K1C+IR group. The 2K1C rats exhibited glomerulosclerosis and tubulointerstitial fibrosis in the unclipped kidney, whereas these changes were attenuated by an iron-restricted diet. Importantly, proteinuria was decreased in the 2K1C+IR group, along with decreased urinary 8-hydroxy-2'-deoxyguanosine excretion and superoxide production of the unclipped kidney. Moreover, the expression of nuclear mineralocorticoid receptor in the unclipped kidney of the 2K1C rats was attenuated by iron restriction. These data indicate a novel effect of iron restriction on hypertension and renal damage in renovascular hypertension.

Iron is associated with the development of hypoxia-induced pulmonary vascular remodeling in mice.

Several recent observations provide the association of iron deficiency with pulmonary hypertension (PH) in human and animal studies. However, it remains completely unknown whether PH leads to iron deficiency or iron deficiency enhances the development of PH.In addition, it is obscure whether iron is associated with the development of pulmonary vascular remodeling in PH. In this study, we investigate the impacts of dietary iron restriction on the development of hypoxia-induced pulmonary vascular remodeling in mice. Eight- to ten-week-old male C57BL/6J mice were exposed to chronic hypoxia for 4weeks. Mice exposed to hypoxia were randomly divided into two groups and were given a normal diet or an iron-restricted diet. Mice maintained in room air served as normoxic controls. Chronic hypoxia induced pulmonary vascular remodeling, while iron restriction led a modest attenuation of this change. In addition, chronic hypoxia exhibited increased RV systolic pressure, which was attenuated by iron restriction. Moreover, the increase in RV cardiomyocyte cross-sectional area and RV interstitial fibrosis was observed in mice exposed to chronic hypoxia. In contrast, iron restriction suppressed these changes. Consistent with these changes, RV weight to left ventricular+interventricular septum weight ratio was increased in mice exposed to chronic hypoxia, while this increment was inhibited by iron restriction. Taken together, these results suggest that iron is associated with the development of hypoxia-induced pulmonary vascular remodeling in mice.