In USA more than 12.5% of all infants are born preterm. Approximately 75% of all perinatal deaths occur among these preterm infants. Preterm infants are frequently very low in birth weight (VLBW) and receive multiple red blood cell (RBC) transfusions. These transfusions pose increased risk of infections and other complications. Since erythropoietin (EPO) stimulates RBC production, EPO treatment of VLBW infants has received attention as a modality for reducing transfusions in this group. The overall hypothesis of this work is that treatment optimization of EPO of anemia in preterm infants requires a comprehensive knowledge of the behavior of RBC and the pharmacokinetic/pharmacodynamics (PK/PD) relationship between EPO and erythropoiesis. Under that overall hypothesis, the specific aims were: 1) To describe erythropoiesis dynamics in preterm infants, 2) To determine and explain the variability in the response to EPO in preterm infants, 3) To evaluate newborn sheep as an experimental model for erythropoiesis in preterm infants, 4) To test the hypothesis that RBC lifespan is shortened under acute hypoxic stress conditions, 5) To test the hypothesis that EPO receptor (EPOR) pool size increases under hypoxic stress conditions and the change in EPOR pool size can be predicted using EPO clearance measurements, 6) To describe the effect of EPOR pool size changes on erythropoiesis kinetics. A model that describes erythropoiesis dynamics in preterm infants as a function of the plasma EPO concentration is presented in Chapter 2. In Chapter 3, several covariates are tested for their ability to identify infants with good EPO responsiveness. The lamb is also tested as an animal model for the erythropoiesis in preterm infants (Chapter 4). In Chapters 5-7, the effect of hypoxic stress conditions on RBC survival was explored defining the relation between the efficacy of EPO and survival of RBC produced as a result of EPO administration. RBC lifespan measurement methods are reviewed in Chapter 5. In Chapter 6, a new methodology for the measurement of RBC lifespan under stress conditions is developed. This new methodology is applied in Chapter 7 to explore the effect of hypoxic stress conditions on the survival of RBC. The study presented in Chapter 8 is undertaken to investigate changes in both EPOR pool size and EPO clearance under hypoxic conditions. An erythropoiesis model that accounts for change in the EPOR pool size under stress conditions is presented in Chapter 9. Analysis of erythropoiesis dynamics in preterm infants demonstrated that a three fold increase in the amount of RBC produced by preterm infants is possible by EPO administration. This emphasizes the potential of using EPO for the management of anemia in preterm infants. Covariate screening identified gestational age as a potential marker for the responsiveness to EPO treatment. PD analysis results in lambs demonstrated similarities between lambs and preterm infants in different erythropoietic characteristics such as sensitivity to EPO in producing RBC, Hb production rate before birth and blood volume. Survival analysis demonstrated that RBC lifespan is not shortened under acute hypoxic conditions Analysis of EPOR mRNA level demonstrated an up regulation of EPOR level under stress conditions accompanied by a parallel increase in EPO clearance. EPOR up regulation under stress conditions level was incorporated in a PD model presented in Chapter 9. The developed model provides a framework for optimizing EPO dosing. Accordingly, an optimal dosing strategy should in general maximize the interaction between EPO and EPOR. Specifically, EPO should be administered when the number of EPOR are close to maximally up-regulated.

Erythroid terminal differentiation couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. I used Epor−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. I found that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. Specifically, I found that high levels of EpoR signaling increase the size and shorten the cycle of early erythroblasts, which are amongst the fastest cycling somatic cells. I confirmed the effect of erythropoietin (Epo) on red cell size in human volunteers, whose mean corpuscular volume (MCV) increases following Epo administration. Our work shows that EpoR signaling alters the expected inverse relationship between cell cycle length and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress. The ability of EpoR signaling to increase cell size in rapidly cycling early erythroblasts suggests that these cells have exceptionally efficient EpoR-driven mechanisms for growth. I found evidence for this in ongoing work, where Epor−/− and Stat5−/− single-cell transcriptomes show dysregulated expression of ribosomal proteins and rRNA transcription and processing genes. Global rates of ribosomal rRNA transcription and protein synthesis increase in an EpoR dependent manner during a narrow developmental window in early ETD, coincident with the time of cell cycle shortening. Our work therefore suggests EpoR-driven regulation of ribosome biogenesis and translation orchestrating rapid cycling and cell growth during early ETD.

No description available.

No description available.

Submitted in partial fulfillment of the requirements for the degree of Doctor In Technology (Clinical Technology), Durban University of Technology, Durban, South Africa, 2016. / Erythropoietin (EPO) is widely used in patients with chronic renal failure and is a necessity. However, due to the cost implications and the medical complications in our population it is imperative to review the factors affecting the process of erythropoiesis and the analysis of cell proliferation and cell viability in the bioassay. Complications such as hypertension and risk of worsening a malignancy cannot be ignored. We had previously analysed variations of erythropoietin levels in haemodialysis patients over a six month period. This study aims to evaluate erythropoiesis in conjunction with various laboratory, demographic, clinical parameters and inflammatory markers, in the population of haemodialysis patients. EPO, antibody level and antibody activity were analysed in the population groups as EPO responsive and EPO sensitive patients. This is a prospective, experimental and controlled study. Fifty nine patients were randomly selected from haemodialysis units of Addington and King Edward VIII Hospitals following an informed consent and 15 healthy individuals were also selected as controls. Demographic parameters (age, sex), clinical parameters (weight, height, skin folding, EPO doses and blood pressures (BP) were recorded. Pre-dialysis serum was used to measure laboratory markers (haemoglobin, transferrin, ferritin, albumin, ESR, C reactive protein, creatinine and urea). EPO levels and antibody levels were measured by ELISA, the optical density of each well was determined within fifteen minutes using the microplate reader set at 450 nm. All results were statistically analysed using SPSS statistical package version 21 (IBMR). Patients requiring very high doses of EPO to reach Hb of 11g/dL, and they remained anaemic after at least three months of adequate EPO doses were considered to be EPO resistant. Those who responded to the usual EPO doses were labelled EPO sensitive. The bioassay was used to quantify cell proliferation and cell viability in the presence of EPO. The UT 7 cells were cultured in medium, in the presence of serum from the EPO resistant, EPO sensitive patients and the healthy, control subjects. Luminescence was read with the Glorunner Microplate Luminometer and was recorded in relative light units (RLU). The analysis revealed: a non-significant positive correlation between haemoglobin and erythropoietin levels. However, a strong negative correlation was found between CRP and albumin level (R= -0.591; (p=0.001), which was not significant. No correlation was found between haemoglobin or erythropoietin levels and CRP or albumin. There was a positive correlation with systolic and diastolic blood pressures and mean arterial pressures which was statistically significant (p <0.05). EPO dosages and Hb levels were correlated significantly (p < 0.05). No correlation of EPO levels and Hb; age and Hb was found to be significant (p = 0.08). The UT 7 cells cultured in serum in medium alone with RHuEPO containing cells were statistically significant (p <0.01)). Reduction of ATP stimulation between medium and serum was observed. However, mean arterial pressures had a significant association with EPO resistance (p = 0.041) odd ratio- 1.066. In conclusion, EPO level is not a useful tool for the monitoring of its use as it does not correlate with EPO goal of red blood production in our patients. The neutralizing antibodies did not correlate with any of our variables contributing to erythropoiesis, and are therefore not confirmed as playing a major role in erythropoiesis. From the analysis of our results the key contributing factors of EPO doses, malnutrition and age were more significant in erythropoiesis. However the higher doses of EPO significantly increased the blood pressures and the mean arterial pressures (MAP). The analysis of the bioassay showed lack of difference between EPO responsive and EPO sensitive patients. This observation warrants further studies to clarify the role of serum of haemodialysis patients in erythropoiesis. / D

Hintergrund Sowohl die Anzahl der Patienten, die an Diabetes mellitus Typ II erkrankten als auch die Zahl der Patienten, die sich einer Dialysetherapie unterziehen mussten, stiegen in den letzten Jahren stetig an. Im Vergleich zur Normalbe-völkerung ist die Mortalität bei Dialysepatienten um ein Vielfaches erhöht. Da mit dem Verlust der Nierenfunktion nicht nur die exkretorischen sondern auch die inkretorischen Aufgaben der Nieren, wie beispielsweise die Ausschüttung des Hormons Erythropoie-tin, nicht mehr geleistet werden können, sind diese Patienten auf eine Substitution angewiesen. Einige Dialyse-Patienten benötigen allerdings überdurchschnittlich viel Erythropoietin um den Ziel Hb-Wert zu erreichen und zu halten. Man spricht hier von EPO-Resistenz. Zwei kleine observationelle Studien haben im letzten Jahr suggeriert, dass die Gabe von Atorvastatin eine Verbesserung der EPO Resistenz bewirken könnte. Auch ein Zusam-menhang des erreichten Hämoglobinwertes und Inflammation mit einer EPO Resistenz wurde diskutiert10,11 Diese Studien waren allerdings weder verblindet noch an einem ausreichend großen Patienten-Klientel getestet. Das Ziel der vorliegenden Untersuchung lag daher darin, den Einfluss von Statinen auf die EPO-Resistenz in einer großen ran-domisierten Studie zu untersuchen. Des Weiteren wurden Zusammenhänge von EPO-Resistenz mit klinischen Parametern und Endpunkten evaluiert. Methoden Den Untersuchungen lagen die Daten der 4D-Studie („Die Deutsche Diabetes Dialyse Studie“), einer multizentrischen, randomisierten, prospektiven Doppelblind-Studie mit 1255 an Diabetes mellitus erkrankten Dialysepatienten zu Grunde. Die Patienten wur-den in dieser Studie randomisiert, entweder 20 mg Atorvastatin pro Tag oder das ent-sprechende Placebo über einen mittleren Beobachtungszeitraum von 4 Jahren zu erhalten. 10 Alle Patienten wurden weniger als 2 Jahre dialysiert und waren im Alter zwischen 18 und 80 Jahren alt. Nach einer Anlaufperiode von 4 Wochen erfolgte die Randomisierung: 619 Studienteilnehmer wurden auf Atorvastatin- und 636 auf Placebo randomisiert. Der primäre Endpunkt der 4D-Studie bestand aus Tod aufgrund kardialer Ursa-chen, tödlichem oder nicht tödlichem Schlaganfall und nicht tödlichem Myokardinfarkt. Die sekundären Endpunkte waren definiert als Tod durch spezifische kardiale und zerebrovaskuläre Ereignisse, sowie Tod infolge anderer Ursachen. In regelmäßigen Abständen wurden die Teilnehmer unter Anderem auf Komedikation, Körpergewicht, Blutdruck, Puls und diverse hämatologische und biochemische Laborparameter untersucht. Im Speziellen wurde getestet, ob eine Wirkung von Atorvastatin 20 mg auf die EPO Resistenz bei Dialysepatienten mit Diabetes mellitus besteht. Dafür wurden die durch-schnittlichen Hämoglobinwerte, die mittlere EPO-Dosis und der mittlere EPO Resistenz Index für beide Patienten Gruppen über den gesamten Beobachtungszeitraum der Studie dokumentiert und miteinander verglichen. Weiterhin waren die Zusammenhänge zwischen EPO Dosis in Kombination mit Hb-Wert bezogen auf die Gesamtmortalität, kardiovaskuläre Ereignisse, plötzlichem Herztod, Schlaganfall und Myokardinfarkt ein Hauptpunkt der Analyse. Des Weiteren wurde die Assoziation von Hb-Variabilität zu den klinischen Endpunkten und die Beziehung zwischen Inflammation und EPO- Resis-tenz in der 4D-Studie evaluiert. Ergebnisse Obwohl eine deutliche Senkung der Lipidparameter durch Atorvastatin auch in dieser Studie nachgewiesen werden konnte (Atorvastatin-Gruppe von 121 mg/dl auf 72 mg/dl in vier Wochen, Placebo-Gruppe weitgehend unverändert), zeigte sich in der Primärstu-die keine signifikante Reduktion des primären Endpunktes. In den speziellen Analysen zur EPO Resistenz in der vorliegenden Arbeit konnte im Rahmen einer Post-hoc Auswertung der 4D Studiendaten gezeigt werden, dass Statine die EPO Resistenz, sowie EPO-Dosis und Hb-Wert von Dialysepatienten nicht signifikant beeinflussen. In der Atorvastatin-Behandlungsgruppe war gegenüber der Placebo-Gruppe keine Verbesserung dieser 3 Parameter während der 4 jährigen Beobachtungszeit zu verzeichnen. Hohe EPO Dosierungen, insbesondere in Zusammenhang mit nied-rigem Hb waren mit erhöhter Mortalität, kardiovaskulären Endpunkten und plötzlichem Herztod assoziiert. Die EPO-Resistenz, repräsentiert durch den EPO-Resistenz-Index ging mit erhöhten Inflammationswerten einher. Die Hämoglobin-Variabilität über ein Jahr zeigte in unserer Studie allerdings keinen Einfluss auf klinische Endpunkte. Schlussfolgerung Es gilt wissenschaftliche Arbeiten mit kleinen Fallzahlen und observationellem Charak-ter durch randomisierte große Studien kritisch zu beurteilen. Die erhöhte Mortalität und die gesteigerte Rate kardialer Ereignisse und Schlaganfälle bei EPO resistenten Patienten geben Anlass zu erhöhter Vorsicht bei der Gabe hoher EPO-Dosen an Dialysepatienten. Statine üben keinen beweisbaren klinisch bedeutsa-men günstigen Effekt auf die EPO-Resistenz bei diesen Patienten aus. Angesichts des Zusammenhanges zwischen EPO-Resistenz und Inflammation ist der antiinflammatorische Beitrag von Statinen -gemessen an anderen EPO-Resistenz bedingenden Faktoren- möglicherweise relativ zu klein, um sich klinisch bemerkbar zu machen. Weitere Studien zur Erforschung von Risikofaktoren für eine EPO-Resistenz sind daher zwingend notwendig, um weitere Zusammenhänge beleuchten und neue Therapiestrategien etablieren zu können. / Atorvastatin does not improve erythropoietin resistance in hemodialysis patients: results from the 4D study ABSTRACT Background: Statins have been suggested to improve erythropoietin resistance in dialysis patients, but data from randomized clinical studies are missing. Furthermore, little is known about the impact of erythropoietin resistance status on specific cardiovascular outcomes in dialysis patients. Methods: We conducted a post-hoc analysis of the German Diabetes and Dialysis Study (4D Study), a multicenter, randomized, double blind prospective study of 1255 patients with type 2 diabetes mellitus undergoing hemodialysis. Patients were randomly assigned to receive 20mg atorvastatin per day or matching placebo. Erythropoietin resistance status was evaluated every 6 months during a median follow-up of 4 years. By Cox regression analyses, we furthermore determined hazard ratios (HR) for pre-specified, adjudicated endpoints according to baseline erythropoietin resistance status and hemoglobin variability: sudden death (n=160), myocardial infarction (MI, n=200), stroke (n=103), cardiovascular events (CVE; n=469), and all-cause mortality (n=617). Results: Patients had a mean age of 66±8 years (54% male). No differences in hemoglobin, erythropoietin dose requirements, or erythropoietin resistance index were found between the atorvastatin- and the placebo-treated groups for any of the time points analysed during follow-up. Erythropoietin resistance was associated with inflammation, and meaningfully affected adverse outcomes: Patients with Epo doses > 100 IU/week/kg necessary for a sufficient hemoglobin level of 11 g/dl had an adjusted 2.5 fold increased risk of stroke. Similarly, these patients experienced significant, &#8805;70% higher rates of sudden death, combined cardiovascular events and mortality, while the risk of myocardial infarction was not affected. Higher hemoglobin variability did not increase adverse outcomes in the present analysis. Conclusions: This study in the 4D randomised controlled trial did not suggest any effect of atorvastatin in improving responsiveness to EPO therapy in maintenance hemodialysis patients. Resistance to erythropoietin was strongly associated with stroke, sudden death, combined cardiovascular events and mortality. Further studies are needed to reduce erythropoietin resistance in hemodialysis patients. INTRODUCTION Despite an increase in the use of erythropoietin over the last decade, a substantial proportion of patients with chronic kidney disease (CKD) fail to achieve hemoglobin targets as recommended by the international guidelines (1,2). In this setting, the term epo-resistance is used to define the patients who fail to attain the target Hb despite a higher than usual dose of erythropoietin, or who continuously need a higher dose to maintain it. Approximately 10% of patients with CKD would fulfil this definition, but the true incidence of Epo hyporesponsiveness is not precisely known. The excessive administration of Epo in hyporensponsive patients poses an enormous burden on the social communities. It is accompanied by very high costs requiring large budgets, and competing with other patients needs. Most importantly, Epo resistance has been shown to be associated with an increased mortality (3). Therefore, it is important to reduce Epo resistance, with the potential to improve patient outcomes and support social healthcare systems. Chronic kidney disease, and in particular end-stage renal disease (ESRD) has been recognized as an inflammatory state with increased levels of CRP, IL-6 and TNF alfa. Evidence suggests that the inflammatory state increases the resistance to erythropoietin. Important mechanisms involved may be that cytokines could directly inhibit erythropoiesis and promote apoptosis of erythroid precursors (4-7), decrease iron availability for erythropoiesis and upregulate hepcidine. Likewise, statins have been suggested to decrease or attenuate inflammation in ESRD patients, and potentially reduce Epo resistance (8,9). However, the latter studies were small and uncontrolled, and the anti-inflammatory effect of statins did not translate into an improvement of survival in randomized trials (10,11). In this context, there has furthermore been much discussion and controversy about the impact of target haemoglobin and Epo responsiveness on cardiovascular events and survival in dialysis patients. The effect of statins on Epo resistance remains largely unclear, as the role of Epo resistance and target hemoglobin for the different components of cardiovascular events and survival in these patients. Hence, we conducted a post-hoc analysis of the 4D study, a randomized controlled trial to evaluate the effect of cholesterol lowering therapy in 1255 dialysis patients with type 2 diabetes mellitus. Our primary aim was to investigate the effect of atorvastatin 20mg od on ESA responsiveness in the patients receiving ESA therapy. Second, we hypothesized an association between ESA responsiveness and inflammation in dialysis patients. Finally, we assessed the effect of ESA responsiveness on sudden cardiac death, stroke, myocardial infarction, combined cardiovascular events and mortality, as the effect of haemoglobin variability on these endpoints. Statistical Analysis Patient characteristics are presented according to categories of Erythropoietin resistance status. Patients were divided into the following four groups: 1) Hb 8804; 11g/dl, EPO use 8804; 100 IU/kg/week 2) Hb 8804; 11g/dl, EPO use > 100 IU/kg/week, 3) Hb > 11g/dl, EPO use 8804; 100 IU/kg/week, 4) Hb > 11g/dl, EPO use > 100 IU/kg/week. Continuous variables were expressed as mean with standard deviation or median with interquartile range (IQR) as appropriate, and categorical variables were expressed as percentages. In order to investigate the impact of statins on erythropoietin resistance, the mean haemoglobin,the mean EPO dose,and the mean ESA index per patient for the 5 years of study were determined. The darbepoetin dose (ug/kg/wk) was converted to the equivalent EPO dose by multiplying x 200.The ESA index was computed from the EPO dose (U/kg/wk) divided by the Hb (g/dL). A comparison was made of the values for each of these three measurements between the atorvastatin- and placebo-treated groups. Furthermore, haemoglobin, Epo dose, and Esa index values were investigated longitudinally across the 5 years of study including differences from baseline by treatment. Second, the values of Epo dose and ESA index at baseline were compared between patients with different levels of CRP in order to assess the association of Epo resistance with inflammation. Patients were thereby grouped according to a CRP level of < or 8805;5mg/dl, and a level of < or 8805;10mg/dl in a second step. Third, the effect of erythropoietin resistance on clinical outcomes was assessed by Kaplan-Meier estimates for incidences of the pre-specified endpoints grouped by the categories of Erythropoietin resistance status (see above). Furthermore, relative risks were derived from Cox regression analyses, i.e. hazard ratios (HR) and corresponding 95% confidence intervals. The analyses were adjusted for the potential confounders age, gender, duration of diabetes, duration of haemodialysis, time on EPO therapy, co-morbidity, cholesterol level, CRP level, and ferritin. Similarly, in order to assess the impact of hemoglobin variability on clinical outcomes, Kaplan-Meier and Cox regression analyses were performed. The standard deviation for the first 4 hemoglobin values (baseline, 1, 6, and 12 months) was determined and patients grouped according to quartiles of this parameter. Kaplan-Meier plots were constructed for each of the four groups, as were adjusted hazard ratios and 95% confidence intervals calculated. All p-values are reported two-sided. Analyses were performed using SAS version 9.1.3. RESULTS Patient characteristics Between March 1998 and October 2002, a total of 1255 patients were included into the 4D study. The mean follow-up period was 3.96 years (median 4.0 years) on atorvastatin and 3.91 years (median 4.08 years) on placebo. During follow-up, 617 patients died, of whom 160 patients died of sudden cardiac death. Furthermore, a total of 200 patients experienced a fatal or non-fatal myocardial infarction. In the study population (n=1255), the mean (SD) age was 65.7 (8.3) years, and 54% of the patients were male. A total of 81% of the patients received erythropoietin. The mean (SD) baseline hemoglobin level was 10.9 (1.4) g/dl; with no significant difference between the atorvastatin and placebo groups. The baseline patient characteristics are shown in Table 1. Effect of atorvastatin on erythropoietin resistance There was no difference between the atorvastatin and placebo groups in mean haemoglobin, mean Epo dose, and mean ESA index, when the mean values over 5 years were calculated per patient. The respective values were 11.2 (1.0) vs 11.2 (1.1) g/dl for haemoglobin (p=0.99), 83.6 (48.5) vs 86.6 (49.8) IU/kg/week for Epo dose (p=0.33) and 7.6 (4.9) vs 7.9 (5.0) IU/kg/week/Hb for the ESA index (p=0.32). The results of the longitudinal analyses investigating the single timepoints separately across the 5 years of study including differences from baseline by treatment are shown in Figures 1A-C. Overall, the differences were very small and not statistically significant. Association of inflammation with erythropoietin resistance Patients with a CRP level above 5mg/L had higher doses of erythropoietin and a higher ESA index as compared to the patients with a CRP below 5mg/L. The median level of epo dose was 69 IU/kg/week (IQR 45 - 101) in the patients without, and 76 IU/kg/week (IQR 47 - 108) in the patients with inflammation, as defined by the cutoff of the CRP at 5mg/L. Accordingly, the values for the ESA index were 6,4 (IQR 4,1 - 9,5) and 7,1 (IQR 4,4 -10,8), respectingly, the comparison of which indicated a trend for a difference, as reflected by a p-value of 0.09. Further analyses were performed, using a higher cutoff for the presence of inflammation with a CRP level of 10mg/L. The association of epo resistance with inflammation was more pronounced, as reflected by significantly different levels of the ESA index in patients with and without inflammation. The ESA index was 6,4 (IQR 4,1 - 9,6) in patients with a CRP &#8804; 10 mg/L, and 7,2 (IQR 4,5 - 11,1) in patients with a CRP > 10 mg/L (p = 0,034). The doses of erythropoietin were 70 (IQR 45 - 101,5) and 77 (IQR 48-113) IU/kg/week, respectively. Erythropoietin resistance status and risk of adverse clinical outcomes In Kaplan-Meier analyses, the group of patients with a sufficient haemoglobin >11 g/dL and low Epo dose &#8804;100 IU/week/kg showed the best survival and lowest rates of adverse cardiovascular outcomes (Figures 2A-D). When investigating relative risks by Cox regression analyses, these patients were used as the reference group. Compared to those, patients with sufficient hemoglobin >11 g/dl, but higher Epo use > 100 IU/week/kg had an adjusted 2.5 fold higher risk of stroke. Furthermore, the risk of sudden death was increased by 79%, and the risk of combined cardiovascular events by 73%, respectively. Similarly, these patients experiened a 70% higher mortality as compared to the reference group. Of note, low hemoglobin levels also affected outcomes, both with the use of low or high Epo doses. These patients with low hemoglobin levels < 11g/dl showed higher risks of sudden death, combined cardiovascular events and mortality as compared to the reference group. In contrast, myocardial infarction was not affected by neither combination of hemoglobin and Epo use (Table 2). Hemoglobin variability and risk of adverse clinical outcomes Overall, the risks of sudden death, stroke and myocardial infarction did not meaningfully differ between the quartiles of hemoglobin variability. Surprisingly, there was a lower incidence of cardiovascular events in the highest as compared to the lowest hemoglobin variability group, when cardiovascular events were analyzed in the combined endpoint. However, the risk of all-cause mortality did not differ across the quartiles of hemoglobin variability. DISCUSSION This study investigated the effect of statin use on erythropoietin resistance in hemodialysis patients, using data from the randomized controlled German Diabetes and Dialysis Study (4D Study). We have shown in a large group of 1255 patients that atorvastatin did not meaningfully affect erythropoietin resistance, the dose of erythropoietin, and the level of hemoglobin during a median of 4 years follow-up. There was an association between erythropoietin resistance and inflammation, as patients with a higher ESA index had higher levels of C-reactive protein. Furthermore, patients with higher Epo doses necessary for a sufficient hemoglobin level of 11 g/dl had an adjusted 2.5 fold increased risk of stroke. Similarly, these patients experienced significantly higher rates of sudden death, combined cardiovascular events and mortality. Hemoglobin variability did not meaningfully impact on adverse outcomes in the present analysis. - comparison with literature - mechanisms This study had certain limitations. It was a post-hoc analysis within a selected cohort of German patients with type 2 diabetes mellitus on hemodialysis. Therefore, the associations found may not be generalisable to other patient populations. The double-blind treatment and setting of a randomized clinical trial was the major strength of this study. Further important strengths include the specific clinical outcomes to be analyzed. In this context, the long-term follow-up, adequate sample size and high incidence of pre-specified and centrally adjudicated endpoints are further to be mentioned. In conclusion, this post hoc analysis of 1255 patients in the 4D randomised controlled trial did not suggest any effect of atorvastatin in improving responsiveness to EPO therapy in maintenance HD patients. Resistance to erythropoietin was a strong risk factor for stroke, and furthermore increased the risks of sudden death, combined cardiovascular events and mortality. Further studies with novel treatments are needed in order to address the problem of erythropoietin resistance in hemodialysis patients.

Erythropoietin is a human natural hormone which task is to regulate the amount of red blood cells in the body. At Centro de Inmunología Molecular, situated in Havana, erythropoietin is produced by recombinant DNA-technique. The protein is purified through several chromatography steps. Among other things, Centro de Inmunología Molecular uses affinity chromatography and ion exchange chromatography. To both of these chromatographic methods, gel is used as stationary phase. The aim of this study was to investigate and determine parameters for characterising of two gels, this because Centro de Inmunología Molecular have to exchange the gels. The reason for the gel exchange is that the currently used gels will not be manufactured any more. The gel used in the affinity chromatography is Chelating Sepharose Fast Flow and the gel used in the ion exchange chromatography is Q Sepharose Fast Flow. For both of this gels kinetic parameters and isotherm parameters were determined by experiments. The isotherm parameters qmax and Kd were calculated from an adsorption isotherm. To be able to calculate qmax and Kd for both Q Sepharose Fast Flow gel and Chelating Sepharose Fast Flow gel different experiments were made. A kinetic adsorption and an isotherm adsorption were made on each gel. The kinetic adsorption was made in due to find out how long the two different processes were supposed to run and to understand which part of the mass transfer that is controlling the rate. There is no use to let the process to be in progress any longer than until the adsorption ceases. For the Q Sepharose Fast Flow gel this was after 200 seconds. The adsorption with the Chelating Sepharose Fast Flow gel never ceased completely, but after 1000 seconds the adsorption was so slow that it would be no use to continue the process. If the processes continue after the calculated times only money, hours and recourses will be wasted. The data that were achieved was plotted in two different isotherm adsorption models both the Freundlich- and the Langmuir model, this to determine which model that had the best fit. One could see that the Q Sepharose Fast Flow gel was following the model of Langmuir better and because of this the Langmuir equation was used to calculate qmax and Kd. The qmax for the Q Sepharose Fast Flow gel agreed a lot with the value that Centro de Inmunología Molecular had assumed. When it came to the Chelating Sepharose Fast Flow gel, the same kind of plotting was made. But one could see that this time the data was following the model of Freundlich much better. Therefore a calculation of the desired qmax was impossible. Only the value of Kd was calculated. Because the company Centro de Inmunología Molecular still needed the value of qmax an assumption that the gel was following the model of Langmuir was made. qmax was calculated but without any satisfied results. The programs Excel, Statgraphic and Matlab have been used in all calculations. / Uppsatsnivå: C

Bei Patienten mit Diabetes mellitus Typ 2 und chronischer Niereninsuffizienz ist Anämie häufig. Zum Teil ist sie durch ungenügende EPO-Produktion bedingt. Zusätzlich wird die Hämoglobinsynthese, wie bei der Anämie chronischer Krankheiten (anemia of chronic disease, ACD) beschrieben, durch entzündliche Vorgänge unterdrückt. Der Stellenwert endogenen Erythropoietins bei Patienten mit diabetischer Nephropathie und ACD bleibt noch unsicher sowie auch der Zusammenhang zwischen EPO und der Nierenfunktion. Diese Querschnittsanalyse schloss 224 Patienten mit Typ 2-Diabetes in allen Stadien chronischer Niereninsuffizienz (CNI-Stadium 1-5) ein. Das mediane Alter betrug 67 Jahre, 54 % waren männlich und die mediane GFR lag bei 49 ml/min. Gemäß den Definitionen der K/DOQI-Richtlinien waren 41 % der Patienten anämisch. Von der Studie ausgeschlossen wurden wegen der Anämie behandelte Patienten und solche mit Eisenmangel. Prädiktoren der log-transformierten EPO-Spiegel wurden unter Benutzung multivariater linearer Regressionsmodelle ausgewertet. Die univariate und inverse Beziehung zwischen GFR und EPO-Spiegeln (p = 0,009) wurde in der multivariaten Analyse nicht-signifikant. Erhöhtes CRP (p < 0,001), niedriges Ferritin (p = 0,002), kardiovaskuläre Ereignisse in der Vorgeschichte (p = 0,02) und Hypertension (p = 0,04) waren nach Adjustierung für Alter, Geschlecht, Hb, GFR und andere klinische Faktoren unabhängig mit erhöhten EPO-Spiegeln assoziiert. In der untersuchten Population fand sich kein Zusammenhang zwischen EPO-Spiegeln und Hämoglobin. Bei diabetischen Patienten mit chronischer Niereninsuffizienz werden die EPO-Spiegel trotz gleichzeitig niedriger Hämoglobinspiegel vor allem durch Entzündungsparameter und den Eisenstatus vorhergesagt und sind dabei unabhängig von der Nierenfunktion. Deshalb könnte die Anämie bei Patienten mit diabetischer Nephropathie hauptsächlich durch Inflammation entstehen, die zu einem relativen Eisenmangel und einer Resistenz des Knochenmarks gegenüber endogenem EPO führt. / BACKGROUND: Anemia is prevalent in patients with type 2 diabetes mellitus (DM) and chronic kidney disease (CKD) and is caused in part by insufficient erythropoietin (EPO) production. In addition, inflammatory processes also suppress hemoglobin synthesis as described in anemia of chronic diseases (ACD). The role of endogenous EPO in diabetic CKD and in ACD and its relationship to kidney function remain uncertain. METHODS: This cross-sectional analysis included 224 diabetic patients with CKD stages 1 to 5 (median age 67 yrs, 54% male, median GFR 49 ml/min). Anemia was defined as per K/DOQI guidelines and was prevalent in 41% of the patients. Patients treated for anemia and iron deficient patients were excluded. Predictors of log-transformed EPO-levels were evaluated using multivariate linear regression modeling. RESULTS: The univariate and inverse relationship between GFR and EPO-level (p = 0.009) became non-significant in multivariate analyses. Elevated C-reactive protein (p < 0.001), low ferritin (p = 0.002) , history of CVD (p = 0.02) and hypertension (p = 0.04) were independently associated with elevated EPO-level, after adjusting for age, gender, hemoglobin, GFR, and other clinical factors. Hemoglobin was not associated with EPO-level in this population. CONCLUSIONS: In diabetic patients with CKD, elevated EPO-levels were predicted mainly by inflammatory markers and iron status, despite low hemoglobin levels and independent of kidney function. Therefore, anemia in diabetic CKD could be explained in part by inflammation-mediated iron dysregulation and resistance to endogenous EPO.

The aim of this thesis was to determine the relevant dose of arbepoetin-alfa (DA) in patients with lymphoproliferative diseases (LPD) and chemotherapy induced anemia (CIA), to study the clinical impact of intravenous (IV) iron supplementation combined with epoetin beta treatment, to identify factors that might predict hemoglobin (Hb) response to treatment with epoetins and to investigate safety of DA. A dose-finding phase II study was able to assess a reasonable DA dose of 2.25 μg/kg once weekly for the treatment of CIA in patients with LPD. Dose-response trends were observed for the different dose cohorts although not statistically significant for any of the endpoints. However a significantly higher proportion of patients achieved Hb response (increase ≥2 g/dL) in the combined DA groups than in placebo (P<0.001). A larger pivotal phase II trial was performed in a similar setting o confirm that the dose 2.25μg/kg once weekly was appropriate and safe. The proportion of patients achieving Hb response was significantly higher in the DA group (60%) than in the placebo group (18%) (P<0,001) and resulted in higher mean changes in Hb than placebo from baseline, 2.66 g/dl versus 0.69 /dl. Also a significantly lower proportion of patients in the DA group (31%) received RBC tranfusions than in the placebo group (48%). The short-term safety of DA with the tested dose was confirmed. The efficacy of DA was consistent for all end points independent of malignancy type or baseline endogenous erythropoietin serum levels. The correction of moderate anemia in truly iron repleted patients with clinically stable LPD not receiving hemotherapy or RBC transfusions with epoetin beta treatment, with or without IV iron treatment was studied in an open label randomized trial. Also the impact on iron kinetics was assessed. The mean change in Hb concentration from baseline to end of treatment (EOT ) was 2.91 versus 1.50 g/dL respectively (P<0.0001). There was a significant (P<0.0001) difference in mean Hb at EOT between the iron and no-iron groups (13.0 g/dL versus 11.8 g/dL). Hb response was achieved by significantly more patients in the iron group (P=0.0012)than in the no-iron group (93% versus 53%) and the median time to achieve a Hb response was 6 weeks in the iron group compared with 12 weeks in the no-iron group. The mean weekly epoetin dose per patient was statistically significant lower in the iron group at week 13 (P =0.029) and at least 25% lower at EOT. To investigate the long-term safety of DA in cancer patients with CIA four previously published double blind, randomized placebo-controlled phase II -III studies were analysed (n = 1.129). Median durations of progression-free survival and overall survival was comparable between DA and placebo for lung cancer (median follow up 15.8 months), for LPD (median follow up 32.6 months) and in the pooled population (follow up 4 months).

Erythropoietin (EPO) is produced in the kidney and in fetal liver in response to hypoxia as well as to CoCl2. The EPO protein and mRNA can be induced in response to both stimuli in the human hepatoma cell (HCC) lines Hep 3B and Hep G2. An oxygen sensing mechanism in which a ligand dependent conformational change in the heme protein produces H2O2 in respone to either hypoxia or Cobalt has been demonstrated. However, an intriguing question can be raised as to why some HCC sublines, such as Hep G2 and Hep 3B are capable of expressing EPO gene, whereas in other HCC sublines, such as J5 and SK-Hep-I are completely devoid of the ability to express EPO gene. Along this line, does ¡§differentiation status¡¨ of these HCC cells play a pivotal role in regulating the expression of EPO gene? Next in line, how a differentiation-associated upregulation of g-glutemylcysteine synthetase (g-GCS), which tightly regulating the biosynthesis of endogenous glutathione(GSH) can modulate the expression of EPO. The objective of this research project was designed to address all these questions. Reported herein are several lines of evidence to demonstrate that endogenous GSH contents do play a pivotal role in the control and regulation of the expression of EPO gene. Firstly, using a group of five HCC lines with varying degrees of differentiation as the experimental model, we demonstrated that the endogenous GSH contents of these HCC cells were differentially upregulated depending on the degree of differentiation with an order of abundance being Hep G2> Hep 3B> J5> Mahlavu> SK-Hep-I. Coincidently, we also found that g-GCS heavy subunit activities as well as its mRNA correlated precisely with this order. Among these HCC cell lines tested, only two well-differentiated sublines, Hep G2 and Hep 3B expressed EPO gene implying that the latter process was dependent upon GSH and suggested a notion that a threshold level might be required for its optimal reactivation. Secondly, to further obtain the evidence to substantiate this possible role of GSH, we then supplemented to the cell culture media with an excessive quantity of nonlethal N-acetylcysteine for the purpose of reinforcing the endogenous GSH biosynthesis. Interestingly, we found that this manipulation could revert the reactivation of EPO gene in cell lines, such as J5 and SK-Hep-I, in which their EPO gene expressions were ortherwise shut down under a normal circumstance. Finally, we were able to demonstrated using RT-PCR and western blotting that the expression of EPO gene was reverted in GCS30, a SK-Hep-I subline that was permanently transfected with g-GCSh and is capable of overly expressing endogenous GSH. Taken together, we demonstrated herein for the first time that, besides hypoxia and CoCl2, endogenous GSH contents can also act as a positive regulator for the expression of EPO gene. The underlying mechanism of how GSH exerts its action in the regulation of EPO expression awaits further clarification.