The genetics of red blood cell density, a biomarker of clinical severity in sickle cell disease

L’anémie falciforme est l’une des maladies du sang les plus répandues chez l’homme. Les complications liées à la maladie sont systémiques. Influant virtuellement tous les organes du corps, cette affection provoque des crises de douleurs imprévisibles et aigües dont les complications mènent parfois à la mort. Le processus à travers lequel un globule rouge sain prend la forme d’une faucille est bien décrit dans la littérature; sous désoxygénation, l’eau et les solutés se retirent des globules rouges, la concentration d’hémoglobine S augmente et nous donne des globules rouges denses et déshydratés qui par la suite deviennent falciformes. Les traitements d’aujourd’hui sont pour la plupart expérimentaux et coûteux. De plus, leurs efficacités à long terme varient d’un patient à l’autre. Il est donc impératif de trouver un biomarqueur qui est à la fois abordable et qui améliore la santé des malades de façon systématique. La densité des globules rouges est un biomarqueur largement ignoré par la communauté médicale dans le contexte de la drépanocytose. Aborder l’étude de la sévérité de cette maladie en se concentrant sur la densité des globules rouges nous met en position d’identifier des traitements pour réhydrater les érythrocytes et leur rendre leur forme originale de disque biconcave. Plusieurs études cliniques et physiologiques se sont penchées sur ce biomarqueur sans explorer le volet génétique. Nous avons cherché à éclaircir cet aspect en menant une étude d’association pangénomique et en examinant les séquences exomiques d’individus avec des mesures de densité extrême. Notre étude d’association pangénomique n’a pas conduit à la découverte de nouveau loci, probablement parce que la taille de notre échantillon, et donc notre puissance statistique, était limitée. En revanche, à travers notre approche de priorisation, nous avons découvert un marqueur intronique qui contrôle l’expression d’ATP2B4, la protéine principale de transport de calcium dans les hématies. Notre séquençage exomique a identifié deux mutations rares faux-sens chez un même patient; l’une dans ATP1B2, un transporteur de Na+/K+, et l’autre dans SPTB, le gène du β-spectrin. Ces mutations expliqueraient pourquoi ce patient a le pourcentage de densité le plus élevé parmi tous nos patients séquencés, et pourquoi il vit avec plusieurs complications de la maladie. Finalement, nous avons localisé une mutation faux-sens rare chez deux patients avec un indice élevé de densité de globule rouge, dans PIEZO1, le canal ionique mécano-sensitif. La mutation est prédite délétère par deux algorithmes de prédiction de fonction protéique.Sickle cell disease is one of most common blood disorder amongst human. The complications associated with the disease are systemic. They damage virtually all the organs of the body, causing severe, unpredictable pain episodes, which repercussions can eventually lead to death. The process through which a biconcave, healthy red blood cell assumes a crescent-shape is well described in the literature; under deoxygenation, as water and solutes leave erythrocytes the concentration of hemoglobin S increases thus giving us dense dehydrated cells and subsequently sickled cells. Today’s current therapies are for the most part experimental, costly, and vary widely in their long-term effectiveness from patient to patient. There is, therefore, a pressing need, to identify a biomarker that is cost-effective and provides positive health outcomes to patients. The density of red blood cell is a biological indicator largely ignored by the medical community in sickle cell disease. Exploring erythrocytes density can facilitate the development of new therapies by targeting channels to rehydrate cells back to their normal shape. Clinical and physiological characterizations of this phenotype exist in many studies, but the genetic characterization is absent. We attempted to elucidate the genetic underpinning of this phenotype, by conducting a genome-wide scan, and examining the whole-exome sequences of individuals with extreme red blood cell density. Our genome-wide association study did not highlight any new loci due to our limited statistical power reflected by the cohort’s small sample size. However, our prioritization approach highlighted an intronic variant that controls the expression of ATP2B4, the main calcium pump in erythrocytes. Our whole-exome sequencing experiment pointed out two rare missense mutations in the same patient; one in ATP1B2, a Na+/K+ transporter, and the other in SPTB, the β-spectrin gene. These variants could explain why he has the highest measured density of red blood cells amongst all of our sequenced patients, and why this person experiences several of the disease-related complications. Another rare missense mutation in two patients with elevated levels of dense cells was discovered in PIEZO1, the mechanosensitive ion channel. The mutation is predicted to be deleterious by both protein function prediction algorithms

Erythrocytes as Biomarkers of Virus and Bacteria in View of Metal Ion Homeostasis

The erythrocyte contributes to the immune system in several ways. It sequesters interferons, interleukins or chemokines and by binding nucleic acid. It binds virus and bacteria and may deliver bacteria to macrophages for phagocytosis. It may also kill bacteria directly with oxygen. For proper function of the erythrocyte, homeostasis of reactive oxygen species, selenium, metal ions and trace elements is important. Erythrocytes display morphological and metabolic changes in diseases like sepsis, and in several genetic diseases. Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), giving rise to the coronavirus disease 2019 (Covid-19), show many erythrocyte changes as compared to healthy controls. The erythrocyte responds to hemolysins by purinergic signaling leading to hemolysis or phosphatidylserine exposure on the plasma membrane. Phosphatidylserine marks erythrocytes for clearance by spleen macrophages. Regulated erythrocyte cell death, also called eryptosis, can be induced by oxidative stress, pathogen infection, and certain diseases like sepsis. Erythrocytes may, in the future, contribute more to diagnosis based on research and diagnostic technological development

Therapeutic Approaches to Limit Hemolysis-Driven Endothelial Dysfunction: Scavenging Free Heme to Preserve Vasculature Homeostasis

Hemolysis results in the release of hemoglobin and heme into the bloodstream and is associated with the development of several pathologic conditions of different etiology, including hemoglobinopathies, hemolytic anemias, bacterial infections, malaria, and trauma. In addition, hemolysis is associated with surgical procedures, hemodialysis, blood transfusion, and other conditions in which mechanical forces can lead to red blood cell rupture. Free plasma hemoglobin and heme are toxic for the vascular endothelium since heme iron promotes oxidative stress that causes endothelial activation responsible for vasoocclusive events and thrombus formation. Moreover, free hemoglobin scavenges nitric oxide, reducing its bioavailability, and heme favours ROS production, thus causing oxidative nitric oxide consumption. This results in the dysregulation of the endothelium vasodilator:vasoconstrictor balance, leading to severe vasoconstriction and hypertension. Thus, endothelial dysfunction and impairment of cardiovascular function represent a common feature of pathologic conditions associated with hemolysis. In this review, we discuss how hemoglobin/heme released following hemolysis may affect vascular function and summarise the therapeutic approaches available to limit hemolysis-driven endothelial dysfunction. Particular emphasis is put on recent data showing the beneficial effects obtained through the use of the plasma heme scavenger hemopexin in counteracting heme-mediated endothelial damage in mouse models of hemolytic diseases

Die Regulation erythrozytären Überlebens und suizidalen Zelltodes

The life span of erythrocytes is tightly regulated. Therefore, a mechanism is required to remove senescent or damaged erythrocytes without rupture of the cell membrane resulting in the release of hemoglobin which may impair kidney function. The mechanism of suicidal erythrocyte death is called eryptosis and shares similarities with apoptosis of nucleated cells such as exposure of phosphatidylserine at the cell surface, increase in cytosolic Ca2+ concentration, blebbing of the membrane, cell shrinkage and enzymatic degradation of the cytoskeletton. The cell shrinkage of eryptotic cells is mediated by a Ca2+-dependent K+ channel, the Gardos channel. Its activation by an increase in the intracellular Ca2+ concentration results in the efflux of K+, Cl- and osmotically obliged water. Phosphatidylserine-exposing erythrocytes are rapidly engulfed by macrophages equipped with phosphatidylserine receptors and degraded. Excessive eryptosis may lead to anemia, the pathological lack of erythrocytes. The present study was performed to elucidate mechanisms regulating erythrocyte survival and suicidal cell death. First, the functional significance of the Gardos channel for suicidal erythrocyte death and erythrocyte clearance was studied. Furthermore, the protective role of Gardos channels during exposure to hemolytic toxins was elucidated. Both issues were addressed by experiments performed in mice lacking the Ca2+-dependent K+ channel KCa3.1, the Gardos channel, and their wildtype littermates. Using patch-clamp recording, flow cytometry, in vitro hemolysis and a mouse sepsis model, it is shown that Gardos channel activity and Gardos effect delay hemolysis of injured erythrocytes and, thus, prevent the disastrous filtration of released hemoglobin into the renal tubular system. In a further series of experiments, the role of the NO/cGMP pathway, a powerful regulator of the life span of a variety of cells, for erythrocyte survival is investigated. Flow cytometry, Western Blotting, hematological counts, and MRI imaging were used to illustrate by means of a cGKI-deficient mouse model that cGKI is a mediator of erythrocyte survival in vitro and in vivo. Moreover, the participation of the phosphoinositide-dependent kinase PDK1, a key element in the phosphoinositol-3-kinase signalling pathway, which is involved in the regulation of ion channels, transporters, cell volume and cell survival, in the regulation of suicdal erythrocyte death was studied. Experiments performed in hypomorphic mice with some 20% of normal PDK1 acitivity and their wildtype littermates revealed that PDK1 deficiency is associated with decreased Ca2+ entry into erythrocytes and thus with blunted eryptotic effects of oxidative stress, osmotic shock and chloride removal. Finally, the functional significance of host pathogen interactions for suicidal erythrocyte death was investigated. Using flow cytometry, it could be shown that peptidoglycan, a main component of the bacterial cell wall, is a potent stimulus of eryptosis and thereby impairs erythrocyte survival. Peptidoglycan-induced eryptosis may therefore, at least in part, account for anemia observed in patients with bacterial infections.Die Lebensdauer von Erythrozyten ist genau reguliert. Aus diesem Grund ist ein Mechanismus erforderlich, der es erlaubt, alte oder geschädigte Erythrozyten abzubauen, ohne daß die Zellmembran reißt und Hämoglobin freigesetzt wird, was zu akutem Nierenversagen führen könnte. Ein solcher Mechanismus ist der suizidale Erythrozytentod, der Eryptose genannt wird und Ähnlichkeiten zur Apoptose kernhaltiger Zellen aufweist wie zum Beispiel die Externalisierung von Phosphatidylserin auf der Zelloberfläche, die Zunahme der intrazellulären Calciumkonzentration, das Abknopsen von Membranteilen, Zellschrumpfung sowie der enzymatische Abbau des Zytoskeletts. Die Zellschrumpfung eryptotischer Zellen wird durch einen Ca2+-abhängigen K+-Kanal, den Gardos-Kanal, vermittelt. Dessen Aktivierung durch die Erhöhung der intrazellulären Calciumkonzentration führt zum Verlust von K+, Cl- und osmotisch folgendem Wasser. Phosphatidylserin-exponierende Erythrozyten werden zügig von Makrophagen, die über Phosphatidylserinrezeptoren verfügen, phagozytiert und abgebaut. Exzessiv gesteigerte Eryptose kann zu Anämie, dem krankhaften Mangel an roten Blutkörperchen, führen. Ziel der vorliegenden Arbeit war es, Mechanismen aufzuklären, die erythrozytäres Überleben sowie suidzidales Sterben steuern. Zunächst wurde die funktionelle Bedeutung des Gardos-Kanals für den suizidalen Erythrozytentod und die Klärung der roten Blutkörperchen aus dem Blut untersucht. Ferner wurde die protektive Wirkung der Gardos-Kanäle bei Einwirkung hämolytischer Toxine studiert. Beide Fragestellungen wurden durch Experimente an Mäusen, die den Ca2+-abhängigen K+-Kanal KCa3.1 nicht exprimierten, und ihren Wildtyp-Geschwistern angegangen. Durch Messungen mit der Membranfleckklemme, mit dem Durchflußzytometer, durch in vitro Hämolyse und mittels eines Maus-Sepsis-Modells wurde gezeigt, daß die Gardos-Kanal-Aktivität und der Gardos-Effekt die Hämolyse geschädigter Erythrozyten verzögern und dadurch die potentiell schädliche Filtration des hämolytisch freigesetzten Hämoglobins in das Nierentubulssystem unterbinden. In weiteren Experimenten wurde die Rolle des NO/cGMP-Signalweges, eines bedeutsamen Regulators der Lebensdauer verschiedener Zellen, für das erythrozytäre Überleben untersucht. Mittels Durchflußzytometrie, Western Blotting, Blutbildern sowie Kernspintomographie, wurde an einem cGKI-defizienten Maus-Modell gezeigt, daß die cGKI ein Mediator erythrozytären Überlebens in vitro und in vivo ist. Desweiteren wurde die Beteiligung der Phosphoinositid-abhängigen-Kinase PDK1, eines Schlüsselenzyms des Phosphoinositol-3-kinase-Signalweges, der bei der Regulation von Ionenkanälen, Transportern, des Zellvolumens und –überlebens mitwirkt, an der Steuerung suizidalen Erythrozytentodes analysiert. Experimente an hypomorphen Mäusen mit lediglich 20%-iger PDK1-Aktivität und ihren Wildtyp-Geschwistern ergaben, daß PDK1-Defizienz mit vermindertem Calciumeinstrom in Erythrozyten und daher mit abgeschächten eryptotischen Effekten von oxidativem Streß, osmotischem Schock und der Entfernung extrazellulären Chlorids verbunden ist. Schließlich wurde die funktionelle Signifikanz von Wirt-Erregerbeziehungen für suizidalen Erythrozytentod untersucht. Durchflußzytometrisch konnte gezeigt werden, daß Peptidoglykan, ein wesentlicher Bestandteil bakterieller Zellwände, ein potenter Auslöser von Eryptose ist und dadurch erythrozytäres Überleben beeinträchtigt. Zumindest theoretisch könnte daher Peptidoglykan-induzierte Eryptose zur Anämie von Patienten mit bakteriellen Infektionen beitragen

Progressive Splenomegalie und Makrozytose bei KCa3.1-defizienten Mäusen: Die physiologische Bedeutung des Gardos-Kanals im Erythrozyten

Der Gardos-Kanal (KCa3.1) vermittelt den Ca2+-abhängigen Ausstrom von K+ und Cl- (den Gardos-Effekt) im Erythrozyten. Hierdurch kommt es zu einer Hyperpolarisation der Zellmembran und einem osmotisch bedingten Schrumpfen der Zelle. Diesem Ca2+-aktivierten K+-Kanal wird daher eine entscheidende Rolle für die physiologische Volumenregulation in zirkulierenden Erythrozyten, sowie auch für Krankheiten mit einer pathologischen Volumenregulation zugeschrieben. Insbesondere in der Pathogenese der Sichelzellanämie nimmt der Gardos-Kanal eine zentrale Stellung ein, wodurch diesem Kanal ein besonderes Interesse zuteil wird. Hochpotente, selektive Gardos-Kanal-Inhibitoren, wie beispielsweise Senicapoc, werden aktuell in der Therapie der Sichelzellanämie evaluiert. Während die pathophysiologische Bedeutsamkeit des Gardos-Kanals in der Sichelzellanämie und anderen Erkrankungen intensiv untersucht wurde, ist die tatsächliche funktionelle Relevanz von KCa3.1 sowohl für die physiologische Volumenregulation als auch für die Mechanismen der Eryptose (in Anlehnung an die Apoptose kernhaltiger Zellen) unklar. Auch wurden die Folgen einer genetischen Störung der Expression bis dato nicht detailliert untersucht. Die vorliegende Studie erlaubt die Einordung der physiologischen Rolle. Bei einer Störung der genetischen Expression von KCa3.1 in Mäusen entwickeln diese Tiere eine progressive, moderate Splenomegalie und eine milde Makrozytose. Die Erythrozyten dieser Knock-out Mäuse weisen eine erhöhte Fragilität bei hypoosmotischem Stress im Sinne einer gestörten Volumenregulation und eine verminderte Deformierbarkeit bei der Filtration durch kleine Poren auf. Diese funktionellen Defekte führen zu einer erhöhten Sequestration in der Milz, begleitet von einer erhöhten Eisenablagerung, die hierauf mit einer Arbeitshypertrophie in Form einer stetig zunehmenden Splenomegalie reagiert. Weiterhin konnte mit dieser Studie gezeigt werden, dass der Gardos-Kanal eine zentrale Stellung in der physiologischen Volumenregulation im Erythrozyten einnimmt, die bei einem Ausfall nicht durch andere Systeme kompensiert werden kann. Anders als von anderen Autoren postuliert, konnte keine maßgebliche Rolle von KCa3.1 für die Eryptose nachgewiesen werden. Dabei stellt die vorliegende Studie die erste in vivo Studie zu diesem Themenkomplex dar

Distal renal tubular acidosis developments in its diagnosis and pathophysiology

This thesis describes two groups of experiments, both relating to the condition of distal renal tubular acidosis (dRTA). In the first, an alternative diagnostic test of dRTA to the ‘gold standard’ short ammonium chloride (NH4Cl) test was assessed. This was achieved by the simultaneous oral administration of the diuretic furosemide and the mineralocorticoid fludrocortisone to increase distal sodium delivery and a-intercalated cell proton secretion. I evaluated 11 control subjects and 10 patients with known dRTA by giving oral NH4Cl or furosemide/fludrocortisone in random order on separate days. 3 subjects were unable to complete the study due to vomiting after the NH4Cl, however there were no adverse effects with furosemide/fludrocortisone administration. The urine pH decreased to less than 5.3 in the controls with both tests, whereas no patients were able to lower their urine pH below 5.3 with either test. The simultaneous administration of furosemide/fludrocortisone proved to be an easy, effective and well-tolerated alternative to the standard NH4Cl test for the diagnosis of dRTA. The second group were laboratory-based molecular physiology experiments. Anion exchanger 1 (AE1) mediates electroneutral anion exchange across cell membranes. It is the most abundant protein in the red cell membrane, but is also found in the basolateral membrane of renal a-intercalated cells, where it is required for urinary acidification. Point mutations have been described that convert the red cell AE1 into a cation conductance. AE1 mutations can also cause hereditary dRTA. I investigated the properties of four dRTA associated AE1 mutations (R586H, G609R, S613F and G701D) by heterologous expression in Xenopus Laevis oocytes. These mutants proved to be functional anion exchangers, unlike the red cell mutants, but also demonstrated a cation ‘leak’. I found a very large leak property in the G701D mutant, which is prevalent in SE Asia. I hypothesised that this property might confer a survival advantage. I characterised three other AR dRTA-associated AE1 mutants found in SE Asia, S773P, \Delta850 and A858D via similar transport experiments in AE1-expressing Xenopus oocytes. These three SE Asian mutants also had cation leaks of similar magnitude to that seen in G701D, a property that distinguishes them as a discrete group. The clustering of these cation-leaky AE1 mutations to malarious areas of SE Asia suggests that they may confer malaria resistance

Mechanisms of xenobiotic-sensitive apoptotic cell death of erythrocytes

Erythrocytes are similar to nucleated cells in that they undergo suicidal death or eryptosis. Mechanism involved in eryptosis may depend on the activation of Ca2+-sensitive cation channels leading to increase intracellular Ca2+ activity. Enhanced cytosolic Ca2+ concentration stimulates phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis is a physiological process which may have an important contribution in the limitation of erythrocyte survival. Excessive eryptosis due to stress, toxicity, diseases or defective compensatory mechanism may lead to anemia. The present observations determined the role of xenobiotics in the regulation of eryptosis as well as their toxic effects for erythrocytes. The first part of the study explored the mechanisms adopted by different foods born mycotoxins (enniatin A, ochratoxin A and zearalenone) in the triggering of eryptosis. Exposure of erythrocytes for 48 hours to enniatin A (≥2.5µM) significantly increased [Ca2+]i, decreased [ATP]i, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Decreased [ATP]i by glucose depletion for 48 hours was similarly followed by increased [Ca2+]i, decreased forward scatter and annexin-V-binding. Annexin-V-binding was blunted by Ca2+-removal, by the cation channel inhibitor amiloride (1mM), by the protein kinase C inhibitor staurosporine (500nM) but not by the pancaspase inhibitor zVAD (10µM). A 48 hour treatment of erythrocytes with ochratoxin A was followed by significant increase of Fluo3-fluorescencei (≥ 2.5 µM), increase of ceramide abundance (10 µM), decrease of forward scatter (≥ 5 µM) and increase of annexin-V-binding (≥ 2.5 µM). Ochratoxin A exposure slightly but significantly enhanced hemolysis (10 µM). Ochratoxin (10 µM) enhanced erythrocyte adhesion to HUVEC. Removal of extracellular Ca2+ significantly blunted, but did not abrogate ochratoxin A-induced annexin V binding. Similar to enniatin A and ochratoxin A, a 48 h treatment of erythrocytes with zearalenone (≥ 25 µM) was resulted into significant increase of [Ca2+]i, significant decrease of forward scatter, and significant increase of annexin-V-binding. The effect on annexin V binding was significantly blunted in the nominal absence of extracellular Ca2+. Zearalenone stimulates the suicidal erythrocyte death, an effect at least partially due to stimulation of Ca2+ entry. The present findings show that, all three mycotoxin (enniatin A, ochratoxin A and zearalenone) which were previously reported to induce apoptotic cell death in nucleated cells, also under their in vivo plasma concentrations act as potent stimulators of suicidal death of erythrocytes despite the absence of gene expression and mitochondria. The second part of the study explored the mechanisms involved in the eryptosis induced by therapeutically important phytochemicals (withaferin A, oridonin and dicoumarol). For 48 hour, erythrocytes were exposed to the three different phytochemicals with indicated concentrations leveling the range of their in vivo plasma levels. Withaferin A significantly decreased forward scatter (at ≥10 µM withaferin concentration) and increased [Ca2+]i (≥5 µM), ROS-formation (≥10 µM) ceramide-formation (≥10 µM) as well as annexin-V-binding (≥5 µM). Withaferin A treatment was followed by slight but significant increase of hemolysis. Extracellular Ca2+ removal, amiloride, and the antioxidant N-acetyl-L-cysteine significantly blunted withaferin A-triggered annexin-V-binding. Erythrocytes were exposed to oridonin. At concentration (≥25µM) oridonin significantly increased cytosolic Ca2+-concentration, increased ceramide formation, decreased forward scatter and triggered annexin V-binding (the latter in >20% of the erythrocytes). Oridonin did not decrease ATP concentration and hemolysed <5% of erythrocytes. The effects of oridonin on annexin V binding were partially reversed in the nominal absence of Ca2+ and by the addition of amiloride (1mM). Dicoumarol (≥10 µM) after incubation, significantly increased [Ca2+]i, enhanced cation channel activity, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Following exposure to 30 µM dicoumarol, annexin-V-binding affected approximately 15%, and hemolysis 2% of treated erythrocytes. The stimulation of annexin-V-binding by dicoumarol was abrogated in the nominal absence of Ca2+. Collectively the data obtained from these studies reveal a completely novel effect of medicinally important phytochemicals (withaferin A, oridonin and dicoumarol) i.e the regulation of calcium-dependent suicidal death erythrocyte death

Στρεσογόνες επιδράσεις της νεφρικής ανεπάρκειας στα ώριμα ερυθροκύτταρα

Τα ερυθροκύτταρα των ασθενών με χρόνια νεφρική ανεπάρκεια (ΧΝΑ) τελικού σταδίου που υποβάλλονται σε θεραπεία αιμοκάθαρσης παρουσιάζουν σημαντικές μεταβολές τόσο στο αιματολογικό πρότυπο σε σχέση με υγιή άτομα όσο και σε χαρακτηριστικά των ερυθροκυττάρων σε επίπεδο οξειδωτικού στρες, μορφολογίας, ερυθρόπτωσης, πρωτεϊνικής σύστασης καθώς και αλλαγές σε χαρακτηριστικά του πλάσματος. Η ανθεκτικότητα στην ερυθροποιητίνη έχει συσχετισθεί με ερυθροκυτταρικές βλάβες χωρίς όμως να έχει μελετηθεί ενδελεχώς αυτός ο μηχανισμός αλληλεπίδρασης. Όσον αφορά στην αιμοκάθαρση, εκτός από τη σημαντική επίδραση που έχει στο προσδόκιμο ζωής των ασθενών με ΧΝΑ και στην αντιμετώπιση της αναιμίας, έχει σημαντική επίδραση και σε χαρακτηριστικά των ερυθροκυττάρων τα οποία σχετίζονται με την εμφάνιση αναιμίας. Στόχος της παρούσας μελέτης υπήρξε η λεπτομερής ανάλυση σε κυτταρικό επίπεδο των ερυθροκυττάρων και του πλάσματος ασθενών με ΧΝΑ τελικού σταδίου υπό συμβατική αιμοκάθαρση οι οποίοι λαμβάνουν πολύ υψηλές δόσεις ερυθροποιητίνης σε σύγκριση με ασθενείς που λαμβάνουν φυσιολογικές δόσεις, με απώτερο στόχο την εύρεση των τροποποιήσεων που προκύπτουν ως αποτέλεσμα της περίσσειας ερυθροποιητίνης στο αίμα των ασθενών. Επιπλέον, μελετήθηκε η επίδραση μιας σχετικά νέας μεθόδου κάθαρσης, της αιμοδιαδιήθησης, σε σχέση με τη χρόνια εφαρμοζόμενη συμβατική αιμοκάθαρση για την εύρεση των επιδράσεων αυτής της μεθόδου στο βαθμό αναιμίας των ασθενών, μέσα από τη μελέτη του αιματολογικού προτύπου αλλά και χαρακτηριστικών των ερυθροκυττάρων και του πλάσματος. Εκτός από τον κλασσικό αιματολογικό και βιοχημικό έλεγχο, στο πλάσμα μετρήθηκε η ολική αντιοξειδωτική ικανότητα με δοκιμασία αναγωγής τρισθενούς σιδήρου (FRAP assay), η ελεύθερη αιμοσφαιρίνη (αιμόλυση) κατά Harboe καθώς και ο πληθυσμός των ολικών και ερυθροκυτταρικών μικροκυστιδίων με κυτταρομετρία ροής. Στα ερυθροκύτταρα, η μορφολογική εκτίμηση έγινε με Ηλεκτρονική Μικροσκοπία Σάρωσης, η πρωτεϊνική σύσταση της ερυθροκυτταρικής μεμβράνης με ανοσοαποτύπωμα κατά Western, η μέτρηση των ενδοκυττάριων επιπέδων ROS και ιόντων ασβεστίου διενεργήθηκε με φθορισμομετρία και τα επίπεδα εξωτερίκευσης φωσφατιδυλοσερίνης ανιχνεύθηκαν με κυτταρομετρία ροής. Επιπλέον, στα ερυθροκύτταρα πραγματοποιήθηκε μέτρηση της οσμωτικής και μηχανικής ευθραυστότητας. Η άμεση επίδραση του ουραιμικού πλάσματος στα ερυθροκύτταρα μελετήθηκε in vitro με πειράματα ανασύστασης. Ακολούθησε στατιστική επεξεργασία των αποτελεσμάτων και ανάλυση δικτύων. Γενικά, τα ουραιμικά ερυθροκύτταρα χαρακτηρίζονται από παθολογικές μορφολογικές τροποποιήσεις, δείκτες απομάκρυνσης από την κυκλοφορία και αυξημένα επίπεδα κυστιδιοποίησης. Μάλιστα, τα επίπεδα των μη αναστρέψιμων μορφολογικών τροποποιήσεων επηρεάζονται σε κάποιο βαθμό από τα επίπεδα αιμοσφαιρίνης αίματος των ασθενών. Η μειωμένη ανταπόκριση στη θεραπεία με ερυθροποιητίνη σχετίζεται με αυξημένα επίπεδα ουραιμικών τοξινών στο πλάσμα, ανεπάρκεια πρωτεϊνών CD59 και κλαστερίνη, αυξημένη πρόσδεση της υπεροξειρεδοξίνης-2 στη μεμβράνη ενώ η αιμοκάθαρση επιφέρει σημαντική αύξηση των επιπέδων εξωτερίκευσης φωσφατιδυλοσερίνης. Αντίθετα, η ανταπόκριση στη θεραπεία σχετίζεται με παθολογικά υψηλή αιμόλυση, μειωμένη ανθεκτικότητα στο μηχανικό στρες, αυξημένη πρόσδεσηανοσοσφαιρινών G στη μεμβράνη και ανεπάρκεια του μορίου CD47, ενώ η διαδικασία της αιμοκάθαρσης οδηγεί σε σημαντική αναδιαμόρφωση της μεμβράνης. Όσον αφορά στη μελέτη των δύο διαφορετικών μεθόδων κάθαρσης, η χρόνια θεραπεία σε αιμοδιαδιήθηση οδηγεί σε αποτελεσματική αντιμετώπιση της αναιμίας, βελτίωση του οξειδοαναγωγικού δυναμικού των ερυθροκυττάρων και καταστολή της κυστιδιοποίησης σε σχέση με τη συμβατική αιμοκάθαρση. Αντίθετα, οι άμεσες επιδράσεις της αιμοδιαδιήθησης περιλαμβάνουν αύξηση του οξειδωτικού στρες, παθολογική αιμόλυση, αύξηση δεικτών απομάκρυνσης από την κυκλοφορία και εμφάνιση στοματοκυττάρωσης. Συμπερασματικά, το περισσότερο τοξικό ουραιμικό περιβάλλον που χαρακτηρίζει τους μη ανταποκρινόμενους ασθενείς σε σχέση με τους ανταποκρινόμενους, αντιμετωπίζεται εν μέρει από τις αντιοξειδωτικές, αντι-αιμολυτικές και αντι-αποπτωτικές ιδιότητες της ευθροποιητίνης και του ουρικού οξέος. Η αναιμία στους μη ανταποκρινόμενους ασθενείς φαίνεται να σχετίζεται κυρίως με ανεπάρκεια πρωτεϊνών που αναστέλουν την ενεργοποίηση του συμπληρώματος, ενώ στους ανταποκρινόμενους με επιταχυνόμενο φαινότυπο γήρανσης και αυξημένη ερυθροφαγοκυττάρωση. Τέλος, υπάρχουν ενδείξεις για διαφορετικό μηχανισμό κυστιδιοποίησης των ερυθροκυττάρων ανάμεσα στις δύο ομάδες των ασθενών. Αντίστοιχα, η θεραπεία σε αιμοδιαδιήθηση μακροπρόθεσμα οδηγεί σε βελτίωση της αναιμίας και των ερυθροκυτταρικών παραμέτρων σε σχέση με τη συμβατική αιμοκάθαρση. Οι αρνητικές επιπτώσεις που παρατηρούνται μετά το τέλος της συνεδρίας κάθαρσης στην αιμοδιαδιήθηση φαίνεται να σχετίζονται με την απώλεια χρήσιμων αντιοξειδωτικών συστατικών του αίματος ταυτόχρονα με την απομάκρυνση των ουραιμικών τοξινών. Η θεραπεία με αντιοξειδωτικά φάρμακα κατά τη διάρκεια της συνεδρίας ή η χρήση κατάλληλων φίλτρων εμπλουτισμένων σε βιταμίνες θα μπορούσε να αναστείλλει τις αρνητικές επιπτώσεις που έχει, έστω και προσωρινά, η θεραπεία της αιμοδιαδιήθησης.End stage renal disease (ESRD) patients exhibit a baseline profile of blood modifications, developed around a pathophysiological core of anemia, uremia, inflammation and oxidative stress. There is however, a huge inter-patient variability in numerous measures of red blood cell (RBC) and blood homeostasis, related in part to the administration of widely variable erythropoietin doses. Dialysis therapy, on the other hand, exerts additional stimuli on cells. Haemodiafiltration (HDF) is a renal replacement therapy which is based on the principles of diffusion and convection. It is generally thought to be more effective than conventional haemodialysis (cHD) in eliminating uremic toxins and in improving anemia; however, its effects on red blood cell (RBC) physiological features have not been examined in depth. This study assesses the physiological profile of RBCs in patients with ESRD receiving standard or high doses of recombinant human erythropoietin (rhEPO) as well as the effects of hemodiafiltration versus conventional hemodialysis in hematologic profile and RBC physiology. Blood samples from twenty-eight patients under sustained conventional hemodialysis, responsive, or not to standard rhEPO administration and thirty-two patients under regular HDF or cHD treatment were examined for haematological and RBC-related parameters compared to healthy donors. Red blood cells and plasma were studied for RBC morphology, fragility, hemolysis, redox status, removal signaling, membrane protein composition, and microvesiculation, in repeated paired measurements accomplished before and right after each dialysis session. Acute effects of uremic plasma on RBC features were examined in vitro through reconstitution experiments. Overall, the ESRD RBCs were characterized by pathological levels of shape distortions, surface removal signaling, and membrane exovesiculation, but reduced fragility compared to healthy RBCs. Irreversible transformation of RBCs was found to be a function of baseline Hb concentration. The more toxic uremic context in non-responsive patients compared to rhEPO responders was blunted in part by the antioxidant, anti-hemolytic, and anti-apoptotic effects of high rhEPO doses, and probably, of serum uric acid. A selective lower expression of RBC membrane in complement regulators (CD59, clusterin) and of CD47 “marker-of- self” was detected in non-responders and responders, respectively. Evidence for different short-term dialysis effects and probably for a different erythrocyte vesiculation mechanism in rhEPO responsive compared to non-responsive patients was also revealed. Regarding dialysis therapy effects, HDF-group of patients was characterized by better anemic indexes, improved redox potential and suppressed exovesiculation of blood cells compared to cHD-group pre-dialysis. However, HDF was associated with a temporary but acute, oxidative stress-driven adverse effect on RBCs, reflected by appearance of haemolysis, removal signaling and stomatocytosis. Conclusively, deregulation of RBC homeostasis might involve diverse molecular pathways driving erythrocyte signaling and removal in rhEPO non-responders compared to responsive patients. Moreover, HDF has beneficial “long-term” effects over cHD on ESRD in respect to anemia and RBC physiology. The adverse short-term effects of HDF on postdialysis plasma and RBCs, which are probably associated with the effective clearance of natural antioxidant components from the uremic plasma, strongly suggest the use of a parallel antioxidant therapy during the dialysis session

Red blood cells: the immune system’s hidden regulator, investigation into the role of red blood cells in inflammatory cytokine signalling

Red blood cells are the most abundant cell type in mammals, although, they are mostly described as inert carriers of haemoglobin that function only in gas exchange and transport. Evidence is now mounting that these enucleate cells are more complex than previously understood. Studies have reported that red blood cells from healthy individuals regulate immune cell activity and maturation, but red blood cells from inflammatory disease cohorts are dysfunctional. Red blood cells are known to bind a small number of chemokines and have been described as a sink for these molecules, and the loss of this activity is correlated with disease progression. This results of this thesis support a broader role for red blood cells in regulating inflammation by acting as a cytokine buffer and modulating cell activity. The aims and hypotheses of this thesis were founded on the discovery that red blood cells are a major reservoir for the pro-inflammatory cytokine, macrophage migration inhibitory factor (MIF); in fact, they contribute 1000-fold more per millilitre than plasma. Red blood cells were also identified to be a major reservoir of 30 additional cytokines, chemokines, and growth factors. Further investigation showed that red blood cells bind and release significant quantities of these proteins, a function that can be modulated by other cells and by enzyme inhibitors. Incubating red blood cells with a cancer cell line (A549 cells) resulted in the significant increase of eight pro-tumorigenic cytokines in the red blood cell lysates. These primed red blood cells altered the activity of lymphocytes by stimulating the proliferation of T cells compared to controls, and promoted the expression of cell activation markers. This study supports the hypothesis that red blood cells act as a buffer for cytokines through binding and release, and that alterations in red blood cells from cell-to-cell interactions affects the activity of T cells. This thesis proposes that red blood cells have multiple functions and the results have implications for the study of inflammation, the role of red blood cells in diagnostics, and on the development of red blood cell derived therapeutics