Epo Deficiency Research Articles

THE EFFECT OF DIFFERENT IMMUNOSUPPRESSION THERAPY ON ERYTHROPOIETIN PRODUCTION IN RENAL TRANSPLANTATION

51 Anemia in chronic renal failure is due to erythropoietin (Epo) deficiency that is expected to normalize after successful kidney transplantation. We previously studied 40 children with excellent graft function for 12 months post kidney transplantation and found a mean hematocrit (Hct) -2 SD from the mean for age and sex. Human hepatoma (Hep3B) cells were chosen as a model to study the effect of immunosuppression therapy on Epo production. Cyclosporine(CSA), Azathioprine (AZA), FK506, and Mycophenolate Mofetil (MPM), under 21% O2 and 1% O2 (hypoxic) conditions were tested. Epo levels were measured in mU/mg protein ± SEM and compared to baseline conditions. Both CSA(10μg/mL) and AZA (40μg/mL) inhibited Epo production under 21% O2,(p<.03, p<.002) and hypoxic conditions (p<.02, p<.05) respectively. Increased concentration of CSA and AZA (100μg/mL) resulted in greater inhibition. FK506 (.0001 -1μM) and MPM (.01-10μM) did not inhibit Epo production. CSA added to high dose AZA or MPM depressed Epo production further than either agents alone in 21% O2 (p<.002 and p<.007) and 1% O2 (p<.004 and p<.0001). We then measured Hct%, plasma Epo, and ferritin, in 4 pediatric renal transplant recipients, ages 4-8 years, 3 were on CSA and one on FK506 at 1, 2, 3, 6, 9 months post transplantation. Simultaneous blood trough levels for CSA were done (whole blood TDx), and for FK506 (whole blood MEIA). No acute rejection episodes or blood transfusions occurred in any patient. Mean Hct% for all patients pre transplant, and then at 1, 3, 6, 9 months post transplantation were 36%, 33%, 38%, 37%, and 38%. All patients had adequate ferritin levels (range of 30-167 ng/ml) in the first 6 months post transplantation. Mean serum creatinine at 6 months was 0.65±.2 mg/dl. Epo levels were compared to each Hct value according to a Reference Regression Graph. An inverse linear relationship between log Epo levels and Hct(R2 = 0.341, p=.0002) was found that was less steep than for healthy adults, and similar to patients with renal failure. No correlation was found between CSA or FK506 levels and Hct. Conclusion: CSA and AZA suppress in-vitro Epo production while FK506 and MPM appear to have no effect. The Epo levels produced after transplantation is inappropriately low compared to Hct. We speculate that the mild anemia in children post renal transplantation is due to inhibition of renal interstitial Epo production related to immunosuppression therapy, specifically CSA. The effect of FK506 and MFM on Epo production in-vivo needs further study.

A quest for erythropoietin over nine decades.

The major research accomplishments of the author are described from the time of his PhD thesis work on the mechanism of cobalt polycythemia to the present day. His early work on the quest for the cell that produces erythropoietin (Epo) to his current work on oxygen sensing and signal transduction pathways involved in erythropoietin gene expression are reported. He describes his main research interest in the mechanism of cobalt polycythemia between 1954 and 1962 and his research on how hormones such as the glucocorticoids function in the regulation of erythropoiesis (1956-1962). His major findings during this period were the discovery that hydrocortisone and corticosterone stimulated erythropoiesis (1958) and that cobalt increased erythropoietin production in the isolated perfused dog kidney (1961). He describes how he was led astray in some of his early studies on the cells in the kidney that produce erythropoietin, because of the less-developed technology available to him at that time; and how in situ hybridization and other molecular biology techniques enabled him to confirm some of the earlier work in mice by other investigators that interstitial cells in the kidney were the site of production of erythropoietin in the primate. His work in the controversial area of the mechanism of the anemia of end-stage renal disease is described in detail, as it pertains to Epo deficiency and suppressed erythroid progenitor cell response to Epo. He also discusses his recent work on signal transduction pathways (hypoxia, nitric oxide, adenosine, and C kinase) in oxygen sensing and Epo gene expression.

Possible mechanism for the anaemia induced by gentamicin in rats

Getamicin, was administered subcutaneously to 6-week-old male F344/M rats at a dose of 100 mg/kg/day for 3 weeks, and the possible mechanism for gentamicin-induced anaemia was investigated. In the dosing group, the erythrocyte count, haematocrit value and haemoglobin concentration were decreased by 15%-17% compared with the values in the control group at the end of the dosing period. The reticulocyte count and plasma erythropoietin (Epo) level were decreased on days 7 and 14. No evidence of haemorrhage, haemolysis or compensatory erythropoiesis was observed in haematology, blood chemistry, gross pathology, organ weights or histopathology. Erythrocyte morphology and osmotic fragility showed no treatment-related changes. On the other hand, histopathological examination of the kidney revealed necrosis and regeneration of the renal tubules on days 14 and 21, suggesting extensive renal injury. Hypocellularity in the bone marrow was seen on day 21, and slightly decreased extramedullary haematopoiesis in the spleen was seen on day 14. It was considered that Epo production was suppressed because the renal peritubular cells are thought to be the site of Epo production. These results suggest that the major cause of the anaemia induced by gentamicin treatment is Epo deficiency following injury to the kidney, the site of Epo production.

Erythropoietin production in rats with post-ischemic acute renal failure

To study the role of erythropoietin (Epo) in the pathogenesis of anemia in acute renal failure (ARF), organ Epo mRNA was measured by RNase protection assay in rats with ARF induced by a one hour-occlusion of the left renal artery. Hematocrit was significantly decreased two hours, 24 hours and one week after renal artery occlusion. A significant reduction in serum haptoglobin at two hours and an increase in serum LDH at 24 hours indicated that hemolysis was the likely cause of the initial fall in hematocrit. However, despite the reduced hematocrit, serum Epo concentrations were not significantly different from controls, suggesting that the anemia is maintained because of lack of an appropriate Epo response. Right renal Epo mRNA levels were not significantly different in all groups, but Epo mRNA levels in post-ischemic kidneys were 50 to 67% lower than in contralateral kidneys. However, Epo mRNA in the post-ischemic kidney was increased sixfold by acute hemorrhage, a rise comparable to the ninefold increase observed in contralateral kidneys. In ARF rats exposed to 7.5% O2 for four hours, right kidney Epo mRNA increased 200-fold over normoxic levels, to a value similar to sham-operated hypoxic controls. Epo mRNA in the post-ischemic kidney also increased 200-fold, to 50% of the level in the contralateral kidney. Hepatic Epo mRNA levels were elevated to comparable levels in both groups. In this ARF model, mild anemia is associated with relative Epo deficiency. In the post-ischemic kidney, a substantial capacity for Epo production is retained but the sensitivity of the Epo response to blood oxygen availability is significantly reduced.

Dysregulation of melatonin metabolism in chronic renal insufficiency: Role of erythropoietin-deficiency anemia

Chronic renal failure (CRF) is associated with a variety of neurological and endocrine disorders. In this study, we examined the effect of CRF and the associated anemia on circadian variation of pineal hormone, melatonin. Animals were studied six weeks after 5/6 nephrectomy (CRF group, N = 26) or sham operation (control group, N = 28). A group of erythropoietin-treated CRF animals (CRF/EPO, N = 6) was included to discern the possible role of EPO-deficiency anemia. Compared with the normal control group, the CRF group showed a marked attenuation of the nocturnal surge in serum melatonin concentration. In addition, pineal gland melatonin content measured after a 12-hour dark cycle (< or = 2 lux) was significantly depressed in the CRF group when compared to that obtained in the control group. However, the CRF animals exhibited appropriate suppression of serum concentration and pineal tissue melatonin content in response to bright light (> or = 2500 lux). Administration of EPO led to correction of the CRF anemia and a marked improvement of the defective nocturnal rhythm of serum melatonin. Based on our results, experimental CRF is associated with a marked attenuation of the normal nocturnal surge of serum melatonin concentration. Regular EPO administration results in the correction of anemia and substantial reversal of this abnormality suggesting the partial role of EPO deficiency. The possible role of melatonin dysregulation in the pathophysiology of CRF and the potential value of melatonin supplementation in this condition is uncertain and awaits future investigations.

Erythropoietin deficiency and relative resistance cause anaemia in post-renal transplant recipients with normal renal function

Following successful renal transplantation, blood erythropoietin (Epo) levels peak in two phases during the first 2-3 months, and blood haemoglobin/haematocrit (HB/Hct) levels are restored to normal in a period of 2-6 months. However, some transplant recipients continue to remain anaemic in spite of normal graft function and in the absence of recognizable causes. The role of endogenous Epo production in the causation of anaemia in such patients is poorly understood and has been investigated in this study. Twenty-three post-renal transplant recipients with stable normal renal function were studied. Eleven of these patients had normal HB/Hct levels (group 1) and served as control for the rest 12 patients with anaemia (group 2). Patients included in group 2 had no readily recognizable cause for their anaemia. Other laboratory and clinical findings were similar in both groups. Patients with erythrocytosis were excluded. Serum Epo levels were measured in all patients. Five patients in group 2 were treated with recombinant human erythropoietin (rHuEpo) and their erythropoietic response was assessed. rHuEpo was discontinued when the target Hb/Hct levels (lowest normal range) were achieved and the patients were followed up for a further period of 9-12 months. Five patients in group 1 had normal expected serum Epo levels whereas the other six patients had inappropriately high serum Epo levels with respect to their Hb/Hct status suggestive of relative ¿EPO resistance'. Serum Epo levels in all patients except two in group 2 were low indicative of 'Epo deficiency'. The two exceptional patients in group 2 had higher serum Epo levels in the presence of anaemia suggestive of relative ¿Epo resistance'. All five patients treated with rHUEpo responded adequately by achieving normal Hb/Hct levels. Three of them were originally ¿Epo deficient' and they reached target Hb/Hct levels in a mean period of 4 weeks, requiring a mean cumulative rHuEpo dose of 428.3 units/kg. The other two patients with higher initial serum Epo levels, and considered to be ¿Epo resistant' required an average of 11 weeks of treatment and a mean cumulative rHuEpo dose of 1582.5 units/kg, indicating an increased Epo demand. On cessation of therapy the Hb/Hct levels fell in all five patients to pretreatment values in 6 months. There are important variations in the endogenous Epo production in renal transplant patients with normal renal function, the cause of which is not clear. Epo deficiency and relative Epo resistance play a causative role for anaemia in some post-renal transplant recipients with stable normal renal function. They respond adequately to rHuEpo administration.

Gene therapy for long-term expression of erythropoietin in rats.

The injection of recombinant erythropoietin (Epo) is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer, and human immunodeficiency virus infections. The ability to deliver this hormone by gene therapy rather than by repeated injections could provide substantial clinical and economic benefits. As a preliminary approach, we investigated in rats the expression and biological effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat Epo cDNA. Vector-derived Epo secretion caused increases in reticulocytes, with peak levels of 7.8-9.6% around day 10 after implantation. The initial elevation in reticulocytes was followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. Ten control and treated animals showed mean hematocrits of 44.9 +/- 0.4% and 58.7 +/- 3.1%, respectively (P < 0.001), and hemoglobin values of 15.6 +/- 0.1 g/dl and 19.8 +/- 0.9 g/dl, respectively (P < 0.001). There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely down regulated in kidney and absent from liver. These results indicate that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.

Serum erythropoietin levels in gynecologic cancer patients during cisplatin combination chemotherapy

To investigate the participation of erythropoietin (Epo) in anemias induced by cisplatin combined chemotherapy, serum Epo levels were measured by radioimmunoassay on a serial basis in eight patients with gynecologic cancer undergoing this chemotherapy. The data demonstrated that serum Epo levels, the mean level before chemotherapy being 20.1 +/- 6.6 mU/ml, were significantly elevated after the first course of the chemotherapy (52.1 +/- 32.2 mU/ml; P less than 0.05) when anemia was not evident. The serum Epo level continued to increase as the course of chemotherapy advanced, and furthermore, patients with normocytic anemia after a multiple course of this chemotherapy still showed high Epo levels (115.2 +/- 53.5 mU/ml) that were appropriate for given degrees of anemia. It is suggested that cisplatin combined chemotherapy caused the elevated serum Epo levels through an unknown mechanism other than anemia, and that in anemias induced by cisplatin, Epo deficiency is not evident.

Serum erythropoietin levels in gynecologic cancer patients during cisplatin combination chemotherapy

To investigate the participation of erythropoietin (Epo) in anemias induced by cisplatin combined chemotherapy, serum Epo levels were measured by radioimmunoassay on a serial basis in eight patients with gynecologic cancer undergoing this chemotherapy. The data demonstrated that serum Epo levels, the mean level before chemotherapy being 20.1 ± 6.6 mU/ml, were significantly elevated after the first course of the chemotherapy (52.1 ± 32.2 mU/ml; P < 0.05) when anemia was not evident. The serum Epo level continued to increase as the course of chemotherapy advanced, and furthermore, patients with normocytic anemia after a multiple course of this chemotherapy still showed high Epo levels (115.2 ± 53.5 mU/ml) that were appropriate for given degrees of anemia. It is suggested that cisplatin combined chemotherapy caused the elevated serum Epo levels through an unknown mechanism other than anemia, and that in anemias induced by cisplatin, Epo deficiency is not evident.

Insulin-Like Growth Factor I: a Modulator of Erythropoiesis in Uraemic Patients?

Anaemia is a feature almost invariably complicating chronic renal failure. Its pathophysiology is multifactorial but the most important cause is erythropoietin (Epo) deficiency. However, either no relation or even a weakly positive relation generally exists between serum immunoreactive (i) Epo and haematocrit values in uraemic anaemia, whereas in anaemias of non-renal origin the correlation is most often strongly negative. Recent evidence indicates that growth hormone also stimulates erythropoiesis. Moreover, late erythroid progenitor cells (CFU-E) require insulin and/or insulin-like growth factor I (IGF-I) for development in vitro. IGF-I has been shown to have a synergistic action with Epo. We have measured serum iEpo and IGF-I levels in 17 haemodialysis patients with severe hyperparathyroidism (mean +/- SEM serum iPTH, 988 +/- 88 pg/ml). Mean age and duration of dialysis treatment were 46.1 +/- 3.4 and 8.8 +/- 1.0 years respectively. Mean haematocrit and haemoglobin values wer 28.1 +/- 1.7% and 9.39 +/- 0.54 g/dl respectively. Mean serum iEpo and IGF-I levels were 20.3 +/- 4.7 mU/ml and 320 +/- 20 ng/ml respectively (normal values for serum iEpo and IGF-I, 17.9 +/- 6 mU/ml and 91 +/- 23 ng/ml respectively). We found that serum IGF-I concentrations were well correlated with haematocrit values (r = 0.68, n = 15, P less than 0.004) whereas serum iEpo values were not (r = 0.41, n = 12, P = 0.18). IGF-I could therefore be an important factor regulating erythropoiesis in uraemic patients, at least when associated with severe hyperparathyroidism.

Monokines inhibiting erythropoietin production in human hepatoma cultures and in isolated perfused rat kidneys

The blood level of erythropoietin (Epo) is often anomalously low in anemic patients with inflammatory or malignant diseases. Therefore, we studied effects of pure recombinant immunomodulatory peptides on Epo formation in cultures of the human hepatoma cell line, HepG2. Interleukin (IL)-1β, IL-1α, and tumor necrosis factor α lowered Epo production with half-maximal inhibition at 2, 5, and 20 U/ml, respectively. IL-6, transforming growth factor β2 and interferon γ did not inhibit. Furthermore, IL-1β (10 U/ml) proved to block Epo formation in isolated serum-free perfused rat kidneys. Proposedly, monokines play a role in the pathogenesis of Epo deficiency in various diseases.

Serum erythropoietin concentrations in patients with myelodysplastic syndromes

Medullary dyserythropoiesis with reduced production of erythrocytes is an early and consistent feature of myelodysplastic syndromes (MDS). The mechanism underlying the disturbed red cell proliferation and maturation is presently unknown. In order to study the role of erythropoietic growth factors, we determined by radioimmunoassay the serum concentrations of immunoreactive erythropoietin (Epo) in 42 non-transfused patients with primary and secondary MDS. Their median hemoglobin concentration at the time of Epo measurement was 9.1 g/dl (range, 5.7–14.6). Compared with the control group, 83% of the MDS patients had increased serum Epo levels, ranging from 26–4530 mU/ml. Although in the entire patient population an inverse relationship between serum Epo and hemoglobin concentrations was noted ( r = −0.35; p = 0.02), Epo titers differed markedly between patients at comparable degrees of anemia. In 7 patients presenting with a hemoglobin concentration between 5.9 and 11.9 g/dl, excessive elevations of Epo levels (> 500 mU/ml) were found. In contrast to previous observations, serum Epo concentrations were not shown to correlate with the percentage of erythroblasts in the bone marrow. There was, however, a significant relationship between the Epo activity and the degree of medullary dyserythropoiesis, as assessed by morphological criteria ( p < 0.01). From these data we conclude that the anemia in MDS is not due to an endogenous Epo deficiency. The marked variability of Epo production in these disorders is not fully explained by the degree of anemia, but may also reflect inherent abnormalities of the myelodysplastic erythropoiesis.

Erythropoietin Deficiency in Acute Renal Failure

Erythropoietin (Epo) was sequentially measured by radioimmunoassay in 11 patients with acute renal failure (ARF) of varied aetiology. Epo rapidly decreased to a level inappropriately low for the haemoglobin, the reduced Epo value persisting throughout the oliguric phase and for up to 2 weeks after the restoration of apparently normal renal function. Epo values found in ARF were: at referral 18.2 +/- 9.5, mid-oliguria 14.4 +/- 6.8, diuresis 15.6 +/- 5.8, and recovery 25.1 +/- 15.8 mU/ml. Results are compared with 34 patients with end-stage chronic renal failure, 42 with non-renal anaemia, and 96 normal subjects. Epo deficiency alone is an inadequate explanation of the rapid reduction in haemoglobin at the onset of ARF, but would appear to be an important factor in the maintenance of anaemia in prolonged ARF and accounts for the slow increase in haemoglobin following recovery.