The Labile Side of Iron Supplementation in CKD.

The practice of intravenous iron supplementation has grown as nephrologists have gradually moved away from the liberal use of erythropoiesis-stimulating agents as the main treatment for the anemia of CKD. This approach, together with the introduction of large-dose iron preparations, raises the future specter of inadvertent iatrogenic iron toxicity. Concerns have been raised in original studies and reviews about cardiac complications and severe infections that result from long-term intravenous iron supplementation. Regarding the iron preparations specifically, even though all the currently available preparations appear to be relatively safe in the short term, little is known regarding their long-term safety. In this review we summarize current knowledge of iron metabolism with an emphasis on the sources and potentially harmful effects of labile iron, highlight the approaches to identifying labile iron in pharmaceutical preparations and body fluids and its potential toxic role as a pathogenic factor in the complications of CKD, and propose methods for its early detection in at-risk patients.

Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism.

The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein.

MitoNEET (mNT) is an outer mitochondrial membrane target of the thiazolidinedione diabetes drugs with a unique fold and a labile [2Fe-2S] cluster. The rare 1-His and 3-Cys coordination of mNT's [2Fe-2S] leads to cluster lability that is strongly dependent on the presence of the single histidine ligand (His87). These properties of mNT are similar to known [2Fe-2S] shuttle proteins. Here we investigated whether mNT is capable of cluster transfer to acceptor protein(s). Facile [2Fe-2S] cluster transfer is observed between oxidized mNT and apo-ferredoxin (a-Fd) using UV-VIS spectroscopy and native-PAGE, as well as with a mitochondrial iron detection assay in cells. The transfer is unidirectional, proceeds to completion, and occurs with a second-order-reaction rate that is comparable to known iron-sulfur transfer proteins. Mutagenesis of His87 with Cys (H87C) inhibits transfer of the [2Fe-2S] clusters to a-Fd. This inhibition is beyond that expected from increased cluster kinetic stability, as the equivalently stable Lys55 to Glu (K55E) mutation did not inhibit transfer. The H87C mutant also failed to transfer its iron to mitochondria in HEK293 cells. The diabetes drug pioglitazone inhibits iron transfer from WT mNT to mitochondria, indicating that pioglitazone affects a specific property, [2Fe-2S] cluster transfer, in the cellular environment. This finding is interesting in light of the role of iron overload in diabetes. Our findings suggest a likely role for mNT in [2Fe-2S] and/or iron transfer to acceptor proteins and support the idea that pioglitazone's antidiabetic mode of action may, in part, be to inhibit transfer of mNT's [2Fe-2S] cluster.

GLP-1-RA Corrects Mitochondrial Labile Iron Accumulation and Improves ß-Cell Function in Type 2 Wolfram Syndrome.

CONTEXT: Type 2 Wolfram syndrome (T2-WFS) is a neuronal and ß-cell degenerative disorder caused by mutations in the CISD2 gene. The mechanisms underlying ß-cell dysfunction in T2-WFS are not known, and treatments that effectively improve diabetes in this context are lacking. OBJECTIVE: Unraveling the mechanisms of ß-cell dysfunction in T2-WFS and the effects of treatment with GLP-1 receptor agonist (GLP-1-RA). DESIGN AND SETTING: A case report and in vitro mechanistic studies. PATIENT AND METHODS: We treated an insulin-dependent T2-WFS patient with the GLP-1-RA exenatide for 9 weeks. An iv glucose/glucagon/arginine stimulation test was performed off-drug before and after intervention. We generated a cellular model of T2-WFS by shRNA knockdown of CISD2 (nutrient-deprivation autophagy factor-1 [NAF-1]) in rat insulinoma cells and studied the mechanisms of ß-cell dysfunction and the effects of GLP-1-RA. RESULTS: Treatment with exenatide resulted in a 70% reduction in daily insulin dose with improved glycemic control, as well as an off-drug 7-fold increase in maximal insulin secretion. NAF-1 repression in INS-1 cells decreased insulin content and glucose-stimulated insulin secretion, while maintaining the response to cAMP, and enhanced the accumulation of labile iron and reactive oxygen species in mitochondria. Remarkably, treatment with GLP-1-RA and/or the iron chelator deferiprone reversed these defects. CONCLUSION: NAF-1 deficiency leads to mitochondrial labile iron accumulation and oxidative stress, which may contribute to ß-cell dysfunction in T2-WFS. Treatment with GLP-1-RA and/or iron chelation improves mitochondrial function and restores ß-cell function. Treatment with GLP-1-RA, probably aided by iron chelation, should be considered in WFS and other forms of diabetes associated with iron dysregulation.

Iron and oxidative stress in cardiomyopathy in thalassemia.

With repeated blood transfusions, patients with thalassemia major rapidly become loaded with iron, often surpassing hepatic metal accumulation capacity within ferritin shells and infiltrating heart and endocrine organs. That pathological scenario contrasts with the physiological one, which is characterized by an efficient maintenance of all plasma iron bound to circulating transferrin, due to a tight control of iron ingress into plasma by the hormone hepcidin. Within cells, most of the acquired iron becomes protein-associated, as once released from endocytosed transferrin, it is used within mitochondria for the synthesis of protein prosthetic groups or it is incorporated into enzyme active centers or alternatively sequestered within ferritin shells. A few cell types also express the iron extrusion transporter ferroportin, which is under the negative control of circulating hepcidin. However, that system only backs up the major cell regulated iron uptake/storage machinery that is poised to maintain a basal level of labile cellular iron for metabolic purposes without incurring potentially toxic scenarios. In thalassemia and other transfusion iron-loading conditions, once transferrin saturation exceeds about 70%, labile forms of iron enter the circulation and can gain access to various types of cells via resident transporters or channels. Within cells, they can attain levels that exceed their ability to chemically cope with labile iron, which has a propensity for generating reactive oxygen species (ROS), thereby inducing oxidative damage. This scenario occurs in the heart of hypertransfused thalassemia major patients who do not receive adequate iron-chelation therapy. Iron that accumulates in cardiomyocytes forms agglomerates that are detected by T2* MRI. The labile forms of iron infiltrate the mitochondria and damage cells by inducing noxious ROS formation, resulting in heart failure. The very rapid relief of cardiac dysfunction seen after intensive iron-chelation therapy in some patients with thalassemia major is thought to be due to the relief of the cardiac mitochondrial dysfunction caused by oxidative stress or to the removal of labile iron interference with calcium fluxes through cardiac calcium channels. In fact, improvement occurs well before there is any significant improvement in the total level of cardiac iron loading. The oral iron chelator deferiprone, because of its small size and neutral charge, demonstrably enters cells and chelates labile iron, thereby rapidly reducing ROS formation, allowing better mitochondrial activity and improved cardiac function. Deferiprone may also rapidly improve arrhythmias in patients who do not have excessive cardiac iron. It maintains the flux of iron in the direction hemosiderin to ferritin to free iron, and it allows clearance of cardiac iron in the presence of other iron chelators or when used alone. To date, the most commonly used chelator combination therapy is deferoxamine plus deferiprone, whereas other combinations are in the process of assessment. In summary, it is imperative that patients with thalassemia major have iron chelators continuously present in their circulation to prevent exposure of the heart to labile iron, reduce cardiac toxicity, and improve cardiac function.

Labile iron in cells and body fluids: physiology, pathology, and pharmacology.

In living systems iron appears predominantly associated with proteins, but can also be detected in forms referred as labile iron, which denotes the combined redox properties of iron and its amenability to exchange between ligands, including chelators. The labile cell iron (LCI) composition varies with metal concentration and substances with chelating groups but also with pH and the medium redox potential. Although physiologically in the lower µM range, LCI plays a key role in cell iron economy as cross-roads of metabolic pathways. LCI levels are continually regulated by an iron-responsive machinery that balances iron uptake versus deposition into ferritin. However, LCI rises aberrantly in some cell types due to faulty cell utilization pathways or infiltration by pathological iron forms that are found in hemosiderotic plasma. As LCI attains pathological levels, it can catalyze reactive O species (ROS) formation that, at particular threshold, can surpass cellular anti-oxidant capacities and seriously damage its constituents. While in normal plasma and interstitial fluids, virtually all iron is securely carried by circulating transferrin (Tf; that renders iron essentially non-labile), in systemic iron overload (IO), the total plasma iron binding capacity is often surpassed by a massive iron influx from hyperabsorptive gut or from erythrocyte overburdened spleen and/or liver. As plasma Tf approaches iron saturation, labile plasma iron (LPI) emerges in forms that can infiltrate cells by unregulated routes and raise LCI to toxic levels. Despite the limited knowledge available on LPI speciation in different types and degrees of IO, LPI measurements can be and are in fact used for identifying systemic IO and for initiating/adjusting chelation regimens to attain full-day LPI protection. A recent application of labile iron assay is the detection of labile components in intravenous iron formulations per se as well as in plasma (LPI) following parenteral iron administration.

Regional siderosis: a new challenge for iron chelation therapy.

The traditional role of iron chelation therapy has been to reduce body iron burden via chelation of excess metal from organs and fluids and its excretion via biliary-fecal and/or urinary routes. In their present use for hemosiderosis, chelation regimens might not be suitable for treating disorders of iron maldistribution, as those are characterized by toxic islands of siderosis appearing in a background of normal or subnormal iron levels (e.g., sideroblastic anemias, neuro- and cardio-siderosis in Friedreich ataxia- and neurosiderosis in Parkinson's disease). We aimed at clearing local siderosis from aberrant labile metal that promotes oxidative damage, without interfering with essential local functions or with hematological iron-associated properties. For this purpose we introduced a conservative mode of iron chelation of dual activity, one based on scavenging labile metal but also redeploying it to cell acceptors or to physiological transferrin. The "scavenging and redeployment" mode of action was designed both for correcting aberrant iron distribution and also for minimizing/preventing systemic loss of chelated metal. We first examine cell models that recapitulate iron maldistribution and associated dysfunctions identified with Friedreich ataxia and Parkinson's disease and use them to explore the ability of the double-acting agent deferiprone, an orally active chelator, to mediate iron scavenging and redeployment and thereby causing functional improvement. We subsequently evaluate the concept in translational models of disease and finally assess its therapeutic potential in prospective double-blind pilot clinical trials. We claim that any chelator applied to diseases of regional siderosis, cardiac, neuronal or endocrine ought to preserve both systemic and regional iron levels. The proposed deferiprone-based therapy has provided a paradigm for treating regional types of siderosis without affecting hematological parameters and systemic functions.

Nutrient-deprivation autophagy factor-1 (NAF-1): biochemical properties of a novel cellular target for anti-diabetic drugs.

Nutrient-deprivation autophagy factor-1 (NAF-1) (synonyms: Cisd2, Eris, Miner1, and Noxp70) is a [2Fe-2S] cluster protein immune-detected both in endoplasmic reticulum (ER) and mitochondrial outer membrane. It was implicated in human pathology (Wolfram Syndrome 2) and in BCL-2 mediated antagonization of Beclin 1-dependent autophagy and depression of ER calcium stores. To gain insights about NAF-1 functions, we investigated the biochemical properties of its 2Fe-2S cluster and sensitivity of those properties to small molecules. The structure of the soluble domain of NAF-1 shows that it forms a homodimer with each protomer containing a [2Fe-2S] cluster bound by 3 Cys and one His. NAF-1 has shown the unusual abilities to transfer its 2Fe-2S cluster to an apo-acceptor protein (followed in vitro by spectrophotometry and by native PAGE electrophoresis) and to transfer iron to intact mitochondria in cell models (monitored by fluorescence imaging with iron fluorescent sensors targeted to mitochondria). Importantly, the drug pioglitazone abrogates NAF-1's ability to transfer the cluster to acceptor proteins and iron to mitochondria. Similar effects were found for the anti-diabetes and longevity-promoting antioxidant resveratrol. These results reveal NAF-1 as a previously unidentified cell target of anti-diabetes thiazolidinedione drugs like pioglitazone and of the natural product resveratrol, both of which interact with the protein and stabilize its labile [2Fe-2S] cluster.