Renal glomerular and tubular responses to glutaraldehyde- polymerized human hemoglobin

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and volume replacement therapeutics, however, their molecular and cellular effects on the vasculature and different organ systems are not fully defined. Using a guinea pig transfusion model, we examined the renal glomerular and tubular responses to PolyHeme, a highly characterized glutaraldehyde-polymerized human hemoglobin with low tetrameric hemoglobin content. PolyHeme-infused animals showed no major changes in glomerular histology or loss of specific markers of glomerular podocytes (Wilms tumor 1 protein, podocin, and podocalyxin) or endothelial cells (ETS-related gene and claudin-5) after 4, 24, and 72 h. Relative to sham controls, PolyHeme-infused animals also showed similar expression and subcellular distribution of N-cadherin and E-cadherin, two key epithelial junctional proteins of proximal and distal tubules, respectively. In terms of heme catabolism and iron-handling responses, PolyHeme induced a moderate but transient expression of heme oxygenase-1 in proximal tubular epithelium and tubulointerstitial macrophages that was accompanied by increased iron deposition in tubular epithelium. Contrary to previous findings with other modified or acellular hemoglobins, the present data show that PolyHeme does not disrupt the junctional integrity of the renal glomerulus and tubular epithelium, and triggers moderate activation of heme catabolic and iron sequestration systems likely as part of a renal adaptive response

Down Selection of Polymerized Bovine Hemoglobins for Use as Oxygen Releasing Therapeutics in a Guinea Pig Model

Editor's Highlight: The development of hemoglobin-based oxygen carriers (HBOCs) as a replacement for whole-blood transfusions has been impeded by their systemic toxicity. This paper presents data from a series of HBOCs, demonstrating one candidate that meets predetermined safety criteria. This approach may allow the development of an acceptable blood substitute for human us

Incidence of gestational trophoblastic disease in South Korea: a longitudinal, population-based study

Introduction We investigated the rate and longitudinal trends of gestational trophoblastic disease (GTD) incidence in the Republic of Korea between 2009 and 2015 using population-based data. Materials and Methods Data of patients diagnosed with GTD from 2009 to 2015 were obtained from the Health Insurance Review and Assessment Service/National Inpatient Sample (HIRA-NIS) in the Republic of Korea. The HIRA annually provides the HIRA-NIS, a collection of clinical data from over one million people. For each year, the HIRA-NIS extracted records of 13% of patients admitted at any one time during the year and 1% of all remaining patients using the weighted sample method. Results Medical records of 370,117 women with at least one pregnancy (GTD, ectopic pregnancy, abortion, or delivery) were extracted from a total of 4,476,495 records. Of these, 372 episodes of GTD were identified in women with a mean age of 35.4 ± 0.7 years. The incidence rate of GTD was 130 ± 10 cases per 100,000 pregnancies, which was classified as hydatidiform mole (HM), invasive mole, or malignant neoplasm of the placenta with incidence rates of 110 ± 10, 20 ± 0, or 10 ± 0 cases per 100,000 pregnancies, respectively. Incidence of GTD was lowest among women in their late 20 s and early 30 s. Occurrences of HM accounted for 80.3% all GTD cases. Weighted logistic analysis indicated that while age significantly affected the incidence of GTD (odds ratio (OR): 2.46; 95% confidence interval (CI) [1.79–3.37]; P < 0.001), socioeconomic status did not (OR: 1.94; 95% CI [1.0–3.79]; P = 0.05). Conclusions In the Republic of Korea, we observed overall incidence rates of GTD and HM of 1.3 and 1.1 per 1,000 pregnancies, respectively, which are similar to those reported in recent Western population-based studies. We also noted that annual incidence rates of GTD stabilized from 2009 to 2015

Red blood cell metabolism in Rhesus macaques and humans: comparative biology of blood storage

Macaques are emerging as a critical animal model in transfusion medicine, because of their evolutionary similarity to humans and perceived utility in discovery and translational science. However, little is known about the metabolism of Rhesus macaque red blood cells (RBC) and how this compares to human RBC metabolism under standard blood banking conditions. Metabolomic and lipidomic analyses, and tracing experiments with [1,2,3-13C3]glucose, were performed using fresh and stored RBC (sampled weekly until storage day 42) obtained from Rhesus macaques (n=20) and healthy human volunteers (n=21). These results were further validated with targeted quantification against stable isotope-labeled internal standards. Metabolomic analyses demonstrated inter-species differences in RBC metabolism independent of refrigerated storage. Although similar trends were observed throughout storage for several metabolic pathways, species- and sex-specific differences were also observed. The most notable differences were in glutathione and sulfur metabolites, purine and lipid oxidation metabolites, acylcarnitines, fatty acyl composition of several classes of lipids (including phosphatidylserines), glyoxylate pathway intermediates, and arginine and carboxylic acid metabolites. Species-specific dietary and environmental compounds were also detected. Overall, the results suggest an increased basal and refrigerator-storage-induced propensity for oxidant stress and lipid remodeling in Rhesus macaque RBC cells, as compared to human red cells. The overlap between Rhesus macaque and human RBC metabolic phenotypes suggests the potential utility of a translational model for simple RBC transfusions, although inter-species storage-dependent differences need to be considered when modeling complex disease states, such as transfusion in trauma/hemorrhagic shock models

Iron accelerates hemoglobin oxidation increasing mortality in vascular diseased guinea pigs following transfusion of stored blood

Non-transferrin-bound iron (NTBI) and free hemoglobin (Hb) accumulate in circulation following stored RBC transfusions. This study investigated transfusion, vascular disease, and mortality in guinea pigs after stored RBC transfusion alone and following cotransfusion with apo-transferrin (apo-Tf) and haptoglobin (Hp). The effects of RBC exchange transfusion dose (1, 3, and 9 units), storage period (14 days), and mortality were evaluated in guinea pigs with a vascular disease phenotype. Seven-day mortality and the interaction between iron and Hb as cocontributors to adverse outcome were studied. Concentrations of iron and free Hb were greatest after transfusion with 9 units of stored RBCs compared with fresh RBCs or stored RBCs at 1- and 3-unit volumes. Nine units of stored RBCs led to mortality in vascular diseased animals, but not normal animals. One and 3 units of stored RBCs did not cause a mortality effect, suggesting the concomitant relevance of NTBI and Hb on outcome. Cotransfusion with apo-Tf or Hp restored survival to 100% following 9-unit RBC transfusions in vascular diseased animals. Our data suggest that increases in plasma NTBI and Hb contribute to vascular disease-associated mortality through iron-enhanced Hb oxidation and enhanced tissue injury

Reversal of hemochromatosis by apo-transferrin in non-transfused and transfused Hbbth3/+ (heterozygous b1/ b2 globin gene deletion) mice

Intermediate beta-thalassemia demonstrates a broad spectrum of sequelae and may require occasional blood transfusions over a lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron (NTBI) in plasma. The iron scavenger and transport protein transferrin (Tf) is a potential treatment being studied for correction of anemia. However, Tf may also function to prevent or reduce iron loading of tissues when exposures to NTBI increase. Here we evaluate the effects of apoTf dosing on tissue iron loading and early tissue pathology in non-transfused and transfused Hbbth3/+ mice. The murine Hbbth3/+ phenotype demonstrates mild to moderate anemia and exhibits consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Our results confirmed that chronic apoTf administration resulted in normalization of anemia. Furthermore, we demonstrate normalization of tissue iron content in liver, kidney and heart and attenuation of early tissue changes in non-transfused Hbbth3/+ mice. ApoTf treatment was also found to attenuate transfusion mediated increases in plasma NTBI and asscociated excess tissue iron loading. These therapeutic effects were associated with normalization of Tf saturation and suppressed plasma NTBI. ApoTf treatment was found to modulate a fundamental iron regulatory pathway as evidenced by decreased erythroid Fam132b (erythroferrone) expression, increased liver HAMP expression and plasma hepcidin-25 lelvels and consequently reduced intestinal ferroportin-1 in apoTf treated thalassemic mice

Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics

BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200-900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions. RESULTS: In the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies. CONCLUSIONS: We present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions

Isolated Hb Providence β82Asn and β82Asp Fractions Are More Stable than Native HbA₀ under Oxidative Stress Conditions

We have previously shown that hydrogen peroxide (H₂O₂) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H₂O₂ when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its β-mutant fractions with HbAo. Relative to HbAo, both βK82N and βK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H₂O₂. However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of β-subunits, and α–α dimer formation in the presence of excess H₂O₂. Mass spectrometric and amino acid analysis revealed that βCys93 and βCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in βK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H₂O₂ and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle

Human hp1-1 and hp2-2 phenotype-specific haptoglobin therapeutics are both effective in vitro and in Guinea pigs to attenuate hemoglobin toxicity

Aims: Infusion of purified haptoglobin (Hp) functions as an effective hemoglobin (Hb) scavenging therapeutic in animal models of hemolysis to prevent cardiovascular and renal injury. Epidemiologic studies demonstrate the phenotype heterogeneity of human Hp proteins and suggest differing vascular protective potential imparted by the dimeric Hp1-1 and the polymeric Hp2-2. Results: In vitro experiments and in vivo studies in guinea pigs were performed to evaluate phenotype-specific differences in Hp therapeutics. We found no differences between the two phenotypes in Hb binding and intravascular compartmentalization of Hb in vivo. Both Hp1-1 and Hp2-2 attenuate Hb-induced blood pressure response and renal iron deposition. These findings were consistent with equal prevention of Hb endothelial translocation. The modulation of oxidative Hb reactions by the two Hp phenotypes was not found to be different. Both phenotypes stabilize the ferryl (Fe(4+)) Hb transition state, provide heme retention within the complex, and prevent Hb-driven low-density lipoprotein (LDL) peroxidation. Hb-mediated peroxidation of LDL resulted in endothelial toxicity, which was equally blocked by the addition of Hp1-1 and Hp2-2. Innovation and Conclusion: The present data do not provide support for the concept that phenotype-specific Hp therapeutics offer differential efficacy in mitigating acute Hb toxicity. Antioxid. Redox Signal. 19, 1619-1633