Anemic Belgrade Research Articles

Iron handling and gene expression of the divalent metal transporter, DMT1, in the kidney of the anemic Belgrade (b) rat

We have previously shown that the rat kidney reabsorbs metabolically significant amounts of iron and that it expresses the divalent metal transporter 1, DMT1. The Belgrade (b) rat carries a mutation in DMT1 gene, which causes hypochromic, microcytic anemia due to impaired intestinal iron absorption and transport of iron out of the transferrin cycle endosome. In the duodenum of b/b rats, expression of DMT1 mRNA and protein is increased, suggesting a feedback regulation by iron stores. The aim of this study was to investigate iron handling and DMT1 expression in the kidneys of Belgrade rats. Animals were maintained for 3 weeks on a synthetic diet containing 185 mg/kg iron (FeSO4), after which functional and molecular parameters were analyzed in male heterozygous (+/b) and homozygous (b/b) rats (N = 4 to 6 for each group). Serum iron concentration was significantly higher in b/b compared to +/b rats while urinary iron excretion rates were unchanged in b/b compared to +/b rats. Northern analysis using a rat DMT1 probe showed comparable mRNA levels between +/b and b/b animals. Western analysis and immunofluorescence microscopy performed using a polyclonal antibody against rat DMT1 showed that DMT1-specific immunoreactivity was almost absent in the kidneys of b/b rats compared to that seen in +/b animals. Our results indicate that the G185R mutation of DMT1 causes protein instability in the kidneys of b/b rats. Given that +/b and b/b rats excrete comparable amounts of iron, the lack of DMT1 protein is compensated by an alternative, yet to be identified, mechanism.

Functional properties of transfected human DMT1 iron transporter.

Recently, mutation of the DMT1 gene has been discovered to cause ineffective intestinal iron uptake and abnormal body iron metabolism in the anemic Belgrade rat and mk mouse. DMT1 transports first-series transition metals, but only iron turns on an inward proton current. The process of iron transport was studied by transfection of human DMT1 into the COS-7 cell line. Native and epitope-tagged human DMT1 led to increased iron uptake. The human gene with the Belgrade rat mutation was found to have one-fifth of the activity of the wild-type protein. The pH optimum of human DMT1 iron uptake was 6.75, which is equivalent to the pH of the duodenal brush border. The transporter demonstrates uptake without saturation from 0 to 50 microM iron, recapitulating earlier studies of isolated intestinal enterocytes. Diethylpyrocarbonate inhibition of iron uptake in DMT1-transfected cells suggests a functional role for histidine residues. Finally, a model is presented that incorporates the selectivity of the DMT1 transporter for transition metals and a potential role for the inward proton current.

Lack of protein 4.1a in red blood cells of the hereditarily anemic Belgrade laboratory (b/b) rat

We have demonstrated that the red blood cell (RBC) membrane of the hereditarily anemic Belgrade laboratory (b/b) rat contains protein 4.1b isoform, only. The evidence are given that the synthesis of protein 4.1 in the b/b rat reticulocytes is the same as in normal rat. When haemolytic anaemia was induced in normal rat by in vivo phenyhydrazine treatment the same phenomenon, i.e., the absence of protein 4.1a in the RBC membrane was observed. The increase of 4.1a isoform was monitored in RBCs during the recovery of normal rat after phenyhydrazine treatment. Hence, the portion of membrane protein 4.1a isoform is increasing during rat RBC aging. Likewise, when the RBC life span is prolonged (but not normalised) in the b/b rats by iron-dextran treatment protein 4.1a is present in small portion in the RBC membrane. All these data indicate that the lack of protein 4.1a isoform in the b/b rat is due to the presence of young RBCs in the circulation. J. Cell. Biochem. 75:56–63, 1999. © 1999 Wiley-Liss, Inc.

Lack of protein 4.1a in red blood cells of the hereditarily anemic belgrade laboratory (b/b) rat.

We have demonstrated that the red blood cell (RBC) membrane of the hereditarily anemic Belgrade laboratory (b/b) rat contains protein 4. 1b isoform, only. The evidence are given that the synthesis of protein 4.1 in the b/b rat reticulocytes is the same as in normal rat. When haemolytic anaemia was induced in normal rat by in vivo phenyhydrazine treatment the same phenomenon, i.e., the absence of protein 4.1a in the RBC membrane was observed. The increase of 4.1a isoform was monitored in RBCs during the recovery of normal rat after phenyhydrazine treatment. Hence, the portion of membrane protein 4.1a isoform is increasing during rat RBC aging. Likewise, when the RBC life span is prolonged (but not normalised) in the b/b rats by iron-dextran treatment protein 4.1a is present in small portion in the RBC membrane. All these data indicate that the lack of protein 4.1a isoform in the b/b rat is due to the presence of young RBCs in the circulation.

Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport.

The Belgrade (b) rat has an autosomal recessively inherited, microcytic, hypochromic anemia associated with abnormal reticulocyte iron uptake and gastrointestinal iron absorption. The b reticulocyte defect appears to be failure of iron transport out of endosomes within the transferrin cycle. Aspects of this phenotype are similar to those reported for the microcytic anemia (mk) mutation in the mouse. Recently, mk has been attributed to a missense mutation in the gene encoding the putative iron transporter protein Nramp2. To investigate the possibility that Nramp2 was also mutated in the b rat, we established linkage of the phenotype to the centromeric portion of rat chromosome 7. This region exhibits synteny to the chromosomal location of Nramp2 in the mouse. A polymorphism within the rat Nramp2 gene cosegregated with the b phenotype. A glycine-to-arginine missense mutation (G185R) was present in the b Nramp2 gene, but not in the normal allele. Strikingly, this amino acid alteration is the same as that seen in the mk mouse. Functional studies of the protein encoded by the b allele of rat Nramp2 demonstrated that the mutation disrupted iron transport. These results confirm the hypothesis that Nramp2 is the protein defective in the Belgrade rat and raise the possibility that the phenotype shared by mk and b animals is unique to the G185R mutation. Furthermore, the phenotypic characteristics of these animals indicate that Nramp2 is essential both for normal intestinal iron absorption and for transport of iron out of the transferrin cycle endosome.

α- And β-Globins of the Anemic Belgrade Laboratory Rat. II. The Effect of Hemin and Iron-Dextran Treatment

We have studied the changes in bone marrow and spleen globin chain levels after in vivo hemin and iron-dextran treatment of hereditarily anemic Belgrade laboratory(b/b) rats. The increase of globin chains was detected in the bone marrow and in the spleen when b/b animals were treated with either iron or hemin. The analysis of changes in α- and β-globin chain ratios revealed the distinctive role of these molecules in regulating globin chain status. Iron-dextran, as expected, ameliorated the imbalance of α- and β-globin chains in the b/b rat spleen. On the other hand, hemin, as we have hypothesized in the accompanying paper, leads to a surplus of β-globin chains in the bone marrow, similar to the one detected in the b/b rat spleen. Therefore, an iron-rich microenvironment has a stimulatory effect, while a hemin-rich microenvironment has an inhibitory effect on erythropoiesis.

α- And β-Globins of the Anemic Belgrade Laboratory Rat. I. Status of α- And β-Globins in Bone Marrow and Spleen

In this study we have demonstrated that the bone marrow of the anemic Belgrade laboratory (b/b) rat, as the primary site of erythropoiesis, has a decreased globin polypeptide synthesis in total protein cell extracts. Therefore, it is the source of red blood cells containing decreased amounts of globin mRNAs and polypeptides. In spite of the fact that the b/b rat shows a splenomegaly, the spleen is not capable of compensating for the anemic state. Spleen erythroid cells are defective in differentiation and contain a decreased share of globin polypeptides in total protein cell extracts compared to the control. Spleen cells are also characterized by a drastic imbalance of α- to β-globin resulting in the β-globin chains surplus.

Evidence for HSP70-like protein in the RBC membrane of the hereditarily anemic Belgrade laboratory (b/b) rat.

We have demonstrated that in normal and b/b rat red blood cells (RBCs) hsp70-like protein (heat shock protein 70-like) is localized in the cytosol and it is exported via exosomes during in vivo reticulocytes maturation. As we have presumed, in the mutant (b/b) rat, hsp70-like protein transfers from cytosol to the RBC membrane. In the normal rat RBCs this happens when those cells are submitted to heat stress conditions. Our study indicates that the presence of hsp70-like protein in the b/b rat RBC plasma membrane is consistent with a primary defect and is not a consequence of life long stress, i.e. hypoxia.

Activity of Antioxidant Defense Enzymes and Glutathione Content in Some Tissues of the Belgrade (b/b) Laboratory Rat

The activity of antioxidant defense (AD) enzymes—superoxide dismutase (SOD, EC 1.15.1.1.), catalase (CAT, EC 1.11.1.6.), glutathione peroxidase (GSH-Px, EC 1.11.1.9.), glutathione-S-transferase (GST, EC 2.5.1.18), glutathione reductase (GR, EC 1.6.4.2) and glutathione (GSH) content of the anemic Belgrade (b/b) laboratory rats—were measured and analyzed in liver, spleen, lung, heart, brain and testes in comparison with nonanemic controls. The activities of hepatic Mn SOD, CAT, GSH-Px and GST ( P < 0.02, P < 0.01 and P < 0.005) were decreased in anemic, comparing with nonanemic animals, whereas the spleen CuZn SOD, Mn SOD, CAT and GSH-Px ( P < 0.005, P < 0.02, P < 0.005 and P < 0.01) activities were increased. In the lung of anemic rats, Mn SOD, GSH-Px and GR ( P < 0.005, P < 0.01, P < 0.05) activities were higher, whereas GST ( P < 0.01) activity was lower in relation to nonanemic ones. In anemic rats, heart Mn SOD ( P < 0.05) activity was increased, brain GSH-Px ( P < 0.005) activity was lower, whereas GR ( P < 0.02) activity was higher compared with nonanemic controls. CuZn SOD ( P < 0.05) activity in the testes was elevated and GSH-Px ( P < 0.05) reduced in anemic animals. GSH content was decreased in the liver ( P < 0.01), lung and brain ( P < 0.005) and increased in the spleen ( P < 0.02) of anemic rats in relation to the controls. Our data suggest phenotype specific differences in the AD system of the Belgrade (b/b) rat tissues in comparison with nonanemic controls.

The seeding efficiency of normal and hereditarily anemic (b/b) rat bone marrow colony forming units-spleen as determined in a "rat to mouse" assay.

We demonstrated recently a decreased number, concentration and proliferative activity of colony forming units-spleen day 8 (CFU-Sd8) in the bone marrow of the hereditarily anemic Belgrade Laboratory (b/b) rat. The main consequence of the genetic defect in the b/b rat is impaired intracellular transport of iron resulting in altered hemopoietic stem and progenitor cell compartments. In this study, the seeding efficiency (f) for normal and b/b rat bone marrow CFU-Sd8 was determined in a "rat to mouse" CFU-S assay. There were no differences in f values for normal and b/b rat bone marrow CFU-Sd8, indicating that the severe anemia and consequent metabolic alterations did not affect f in the b/b rat. The calculation of CFU-Sd8 concentration per 10(5) normal rat bone marrow cells using f as a correction factor revealed a value similar to that found for populations of bone marrow cells defined by phenotypes Ox7+ (20%), Ox22-, W3/13dim, considered to be characteristic of CFU-S. The results revealed the usefulness of the "rat to mouse" assay for the determination of rat hemopoietic stem cells.

The Disbalance of α- and β-Globins in Anemic Belgrade Rat Red Blood Cells

The Belgrade Laboratory (b/b) rat has an autosomal mutation which in homozygous state induces severe anemia. This study was based on solubilization of total rat globin chains and their separation into α- and β-globins using a 20% SDS polyacrilamide gel. These analyses demonstrated that the disbalance of α/β globins in b/b red blood cells (RBC) is due to decreased level of α-globins. Iron-dextran administration corrected the level and globin ratio in b/b RBC thus confirming that the iron deficiency is the primary defect in b/b rats.

Decreased plasma proteins, increased total plasma-free amino acids, and disturbed amino acid metabolism in the hereditary severe anemia of the Belgrade laboratory (b/b) rat.

The plasma amino acid pattern has been investigated in severely anemic Belgrade laboratory (b/b) rats. Nonanemic heterozygous (b/+) or normal homozygous (+/+) rats of the same age (six weeks) were used as controls. Decreased plasma proteins, increased total free amino acid, and urea concentrations in plasma associated with increased urea and 3-methylhistidine urinary excretion were found, indicating protein and amino acid metabolic alterations in anemic b/b rats. Plasma alanine, glutamine, tyrosine, and phenylalanine concentrations were increased. The significantly reduced molar ratio between valine+leucine+isoleucine and phenylalanine+tyrosine suggested severe disturbance in the hepatic energy-producing system and derangement of hepatic energy status. Partial or complete reversal of the anemia within 3 days by red blood cell transfusion or within 3 weeks by iron treatment resulted in normalization of tyrosine, alanine, glutamine, and total amino acid concentrations in plasma, as well as of molar ratio between valine+leucine+isoleucine and phenylalanine+tyrosine. This indicated a better oxygen supply to the liver and normalization of the hepatic energy status. These findings suggest that the metabolic disturbances in the b/b rat are the consequence of hypoxia due to the severe anemia.

A pool of nonpolysomal globin mRNAs in globin deficient reticulocytes of the anemic Belgrade rat

In anemia of the Belgrade rat ( b b ) reticulocytes contain less than half of the normal amount of mRNA for seven adult rat globin chains. cDNA hybridization measurements of the relative sizes of polysomal and nonpolysomal pools of globin mRNAs in these cells show that 45% of all globin mRNA molecules are not used at any given time in protein synthesis. This implies a translational control which ensures a production of globin chains in a correct ratio despite a severe mRNA unbalance.