Iron-Hepcidin Dysmetabolism,Anemia and Renal Hypoxia,Inflammation and Fibrosis in the Remnant Kidney Rat Model

Anemia is a common complication of chronic kidney disease (CKD) that develops early and its severity increases as renal function declines. It is mainly due to a reduced production of erythropoietin (EPO) by the kidneys; however, there are evidences that iron metabolism disturbances increase as CKD progresses. Our aim was to study the mechanisms underlying the development of anemia of CKD, as well as renal damage, in the remnant kidney rat model of CKD induced by 5/6 nephrectomy. This model of CKD presented a sustained degree of renal dysfunction, with mild and advanced glomerular and tubulointerstitial lesions. Anemia developed 3 weeks after nephrectomy and persisted throughout the protocol. The remnant kidney was still able to produce EPO and the liver showed an increased EPO gene expression. In spite of the increased EPO blood levels, anemia persisted and was linked to low serum iron and transferrin levels, while serum interleukin (IL)-6 and high sensitivity C-reactive protein (hs-CRP) levels showed the absence of systemic inflammation. The increased expression of duodenal ferroportin favours iron absorption; however, serum iron is reduced which might be due to iron leakage through advanced kidney lesions, as showed by tubular iron accumulation. Our data suggest that the persistence of anemia may result from disturbances in iron metabolism and by an altered activity/function of EPO as a result of kidney cell damage and a local inflammatory milieu, as showed by the increased gene expression of different inflammatory proteins in the remnant kidney. In addition, this anemia and the associated kidney hypoxia favour the development of fibrosis, angiogenesis and inflammation that may underlie a resistance to EPO stimuli and reduced iron availability. These findings might contribute to open new windows to identify putative therapeutic targets for this condition, as well as for recombinant human EPO (rHuEPO) resistance, which occurs in a considerable percentage of CKD patients.     

Mitochondrial DNA Damage in Iron Overload

Chronic iron overload has slow and insidious effects on heart, liver, and other organs. Because iron-driven oxidation of most biologic materials (such as lipids and proteins) is readily repaired, this slow progression of organ damage implies some kind of biological “memory.” We hypothesized that cumulative iron-catalyzed oxidant damage to mtDNA might occur in iron overload, perhaps explaining the often lethal cardiac dysfunction. Real time PCR was used to examine the “intactness” of mttDNA in cultured H9c2 rat cardiac myocytes. After 3–5 days exposure to high iron, these cells exhibited damage to mtDNA reflected by diminished amounts of near full-length 15.9-kb PCR product with no change in the amounts of a 16.1-kb product from a nuclear gene. With the loss of intact mtDNA, cellular respiration declined and mRNAs for three electron transport chain subunits and 16 S rRNA encoded by mtDNA decreased, whereas no decrements were found in four subunits encoded by nuclear DNA. To examine the importance of the interactions of iron with metabolically generated reactive oxygen species, we compared the toxic effects of iron in wild-type and rho^o cells. In wild-type cells, elevated iron caused increased production of reactive oxygen species, cytostasis, and cell death, whereas the rho^o cells were unaffected. We conclude that long-term damage to cells and organs in iron-overload disorders involves interactions between iron and mitochondrial reactive oxygen species resulting in cumulative damage to mtDNA, impaired synthesis of respiratory chain subunits, and respiratory dysfunction.Patients with primary or secondary iron overload are liable to cardiac and hepatic failure, and type II diabetes. Iron is required for the activity of numerous iron- and heme-containing proteins, but “free” (i.e. redox active) iron catalyzes the formation of highly toxic reactive oxygen species (ROS)² that damage lipids, proteins, and DNA (1). This damage is assumed to arise from iron-catalyzed hydroxyl radical formation or, perhaps more likely, iron-centered radicals such as ferryl and perferryl (2, 3). Iron-driven oxidation events require that the metal interact with cellular oxidizing and reducing equivalents such as superoxide and hydrogen peroxide, a major source of which is “leak” of electrons from the mitochondrial electron transport chain (4–6).The present investigations were focused on the etiology of iron-mediated cardiac damage and specifically on the question of why, in patients with chronic iron overload, damage to organs such as the heart develops over a period of years, whereas most types of iron-mediated oxidation events can be repaired within minutes or hours. We have investigated the hypothesis that cumulative damage to DNA, specifically mtDNA, is critical to the slow development of cardiac dysfunction in chronic iron overload. In partial support of this idea, earlier studies clearly show that iron does promote DNA base oxidation as well as single and double strand DNA breaks. Mitochondrial DNA may be particularly vulnerable to such oxidation events inasmuch as it lacks histones, has less effective repair systems and, perhaps most importantly, resides within an organelle that ceaselessly generates ROS.Here, we report that, in cultured rat cardiac myocytes, iron overload causes (i) progressive loss of intact mtDNA, (ii) decreased expression of respiratory chain subunits encoded by mitochondrial, but not nuclear, DNA, and (iii) diminished respiratory function. Furthermore, it appears that iron-mediated cytotoxicity involves ROS generated by the mitochondrion itself because cells lacking mtDNA (and, therefore, respiration) are remarkably tolerant of iron overload. Overall, our results suggest that the slowly developing cardiac dysfunction seen in chronic iron overload arises secondary to cumulative iron-driven oxidant damage to mtDNA.     

Functional specialization within the Fur family of metalloregulators

The ferric uptake regulator (Fur) protein, as originally described in Escherichia coli, is an iron-sensing repressor that controls the expression of genes for siderophore biosynthesis and iron transport. Although Fur is commonly thought of as a metal-dependent repressor, Fur also activates the expression of many genes by either indirect or direct mechanisms. In the best studied model systems, Fur functions as a global regulator of iron homeostasis controlling both the induction of iron uptake functions (under iron limitation) and the expression of iron storage proteins and iron-utilizing enzymes (under iron sufficiency). We now appreciate that there is a tremendous diversity in metal selectivity and biological function within the Fur family which includes sensors of iron (Fur), zinc (Zur), manganese (Mur), and nickel (Nur). Despite numerous studies, the mechanism of metal ion sensing by Fur family proteins is still controversial. Other family members use metal catalyzed oxidation reactions to sense peroxide-stress (PerR) or the availability of heme (Irr).     

Altered expression of iron transport proteins in streptozotocin-induced diabetic rat kidney

Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague-Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.     

Iron-induced oxidative stress up-regulates calreticulin levels in intestinal epithelial (Caco-2) cells

Calreticulin, a molecular chaperone involved in the folding of endoplasmic reticulum synthesized proteins, is also a shock protein induced by heat, food deprivation, and chemical stress. Mobilferrin, a cytosolic isoform of calreticulin, has been proposed to be an iron carrier for iron recently incoming into intestinal cells. To test the hypothesis that iron could affect calreticulin expression, we investigated the possible associations of calreticulin with iron metabolism. To that end, using Caco-2 cells as a model of intestinal epithelium, the mass and mRNA levels of calreticulin were evaluated as a function of the iron concentration in the culture media. Increasing the iron content in the culture from 1 to 20 microM produced an increase in calreticulin mRNA and a two-fold increase in calreticulin. Increasing iron also induced oxidative damage to proteins, as assessed by the formation of 4-hydroxy-2-nonenal adducts. Co-culture of cells with the antioxidants quercetin, dimethyltiourea and N-acetyl cysteine abolished both the iron-induced oxidative damage and the iron-induced increase in calreticulin. We postulate that the iron-induced expression of calreticulin is part of the cellular response to oxidative stress generated by iron.     

Physiology of iron transport and the hemochromatosis gene

Iron is essential for fundamental cell functions but is also a catalyst for chemical reactions involving free radical formation, potentially leading to oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated to maintain an adequate substrate while also minimizing the pool of potentially toxic "free iron." The main control of body iron homeostasis in higher organisms is placed in the duodenum, where dietary iron is absorbed, whereas no controlled means of eliminating unwanted iron have evolved in mammals. Hereditary hemochromatosis, the prototype of deregulated iron homeostasis in humans, is due to inappropriately increased iron absorption and is commonly associated to a mutated HFE gene. The HFE protein is homologous to major histocompatibility complex class I proteins but is not an iron carrier, whereas biochemical and cell biological studies have shown that the transferrin receptor, the main protein devoted to cellular uptake of transferrin iron, interacts with HFE. This review focuses on recent advances in iron research and presents a model of HFE function in iron metabolism.     

A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo

Free radical formation in heme proteins is recognised as a factor in mediating the toxicity of peroxides in oxidative stress. As well as initiating free radical damage, heme proteins damage themselves. Under extreme conditions, where oxidative stress and low pH coincide (e.g., myoglobin in the kidney following rhabdomyolysis and hemoglobin in the CSF subsequent to subarachnoid hemorrhage), peroxide can induce covalent heme to protein cross-linking. In this paper we show that, even at neutral pH, the heme in hemoglobin is covalently modified by oxidation. The product, which we term OxHm, is a "green heme" iron chlorin with a distinct optical spectrum. OxHm formation can be quantitatively prevented by reductants of ferryl iron, e.g., ascorbate. We have developed a simple, robust, and reproducible HPLC assay to study the extent of OxHm formation in the red cell in vivo. We show that hemoglobin is oxidatively damaged even in normal blood; approximately 1 in 2,000 heme groups exist as OxHm in the steady state. We used a simple model (physical exercise) to demonstrate that OxHm increases significantly during acute oxidative stress. The exercise-induced increase is short-lived, suggesting the existence of an active mechanism for repairing or removing the damaged heme proteins.     

Dietary vitamin E reduces labile iron in rat tissues

Previous studies have shown that dietary vitamin E reduced generation and/or levels of superoxide. As superoxide has potential to release iron from its transport and storage proteins, and labile or available form of iron is capable of catalyzing the formation of reactive hydroxyl radicals, the effect of dietary vitamin E on labile iron pool was studied in rats. One-month-old Sprague-Dawley male and female rats were fed a basal vitamin E-deficient diet supplemented with 0, 20, 200, or 2,000 IU vitamin E/kg diet for 90 days. The levels of labile iron were measured in the liver, kidney, spleen, heart and skeletal muscle. Additionally, the levels of lipid peroxidation products were measured. The results showed that, except for labile iron in the heart of male rats, dietary vitamin E dose dependently reduced the levels of labile iron and lipid peroxidation products in all tissues of male and female rats. The findings suggest that dietary vitamin E may protect against oxidative tissue damage by reducing the generation and/or level of superoxide, which in turn attenuates the release of iron from its protein complexes.     

Mitochondrial ferritin expression in adult mouse tissues.

Mitochondrial ferritin (FtMt) is a novel ferritin type specifically targeted to mitochondria. It is highly expressed in the human testis and in sideroblasts from patients with sideroblastic anemia, but other organs have not been studied. To study its expression in the main organs of the mouse, we first used RT-PCR and then produced recombinant mouse FtMt and specific antibodies. Immunohistochemistry analyses confirmed that FtMt is highly expressed in mouse testis, particularly in spermatocytes and interstitial Leydig cells. The protein was also identified in other organs including heart, brain, spinal cord, kidney, and pancreatic islet of Langerhans but not in liver and splenocytes, which have iron storage function and express high levels of cytosolic ferritins. Results indicate that the primary function of ferritin FtMt is not involved in storing cellular or body iron, but its association with cell types characterized by high metabolic activity and oxygen consumption suggests a role in protecting mitochondria from iron-dependent oxidative damage.     

Proteomic analysis of iron overload in human hepatoma cells

Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.     

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δyfh1 sensitivity to menadione. Finally, a Δsod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.     

铁代谢蛋白在肾脏的表达与功能

最近的研究证实,肾小管细胞具有能力表达包括转铁蛋白受体1(transferrin receptor-1,TfR1)、二价金属离子转运蛋白1(divalent metal transporter-1,DMT1)、膜铁转运蛋白1(ferroportin-1,FPN1)、铁调节蛋白(iron regulatory protein,IRP)和铁调素(hepcidin,Hepc)在内的几乎所有铁代谢蛋白.这些蛋白质的存在以及相关研究显示肾脏可能具有排出多余铁的功能,因此对体铁平衡起有十分重要的作用.     

Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.     

Ferritin,iron homeostasis,and oxidative damage

Ferritin is one of the major proteins of iron metabolism. It is almost ubiquitous and tightly regulated by the metal. Biochemical and structural properties of the ferritins are largely conserved from bacteria to man, although the role in the regulation of iron trafficking varies in the different organisms. Recent studies have clarified some of the major aspects of the reaction between iron and ferritin, which results in the formation of the iron core and production of hydrogen peroxide. The characterization of cellular models in which ferritin expression is modulated has shown that the ferroxidase catalytic site on the H-chain has a central role in regulating iron availability. In turn, this has secondary effects on a number of cellular activities, which include proliferation and resistance to oxidative damage. Moreover, the response to apoptotic stimuli is affected by H-ferritin expression. Altered ferritin L-chain expression has been found in at least two types of genetic disorders, although its role in the determination of the pathology has not been fully clarified. The recent discovery of a new ferritin specific for the mitochondria, which is functionally similar to the H-ferritin, opens new perspectives in the study of the relationships between iron, oxidative damage and free radicals.     

Lipocalin-2 (24p3/neutrophil gelatinase-associated lipocalin (NGAL)) receptor is expressed in distal nephron and mediates protein endocytosis

In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+(7)-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+(7)-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123±50 nM was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+(7)-MT toxicity in distal nephron segments.     

Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1^gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1^gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1^gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1^gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.KEY WORDS: NCU-G1, Lysosome, Fibrosis