Erythropoiesis after kidney transplantation: the role of erythropoietin, burst promoting activity and early erythroid progenitor cells.

Successful kidney transplantation is followed by the anemia correction due to re-establishment of normal erythropoietin secretion. The possible role of growth factors and cytokines regulating hematopoiesis in this anemia correction are not completely understood. The aim of this study was to investigate the role of erythropoietin and other stimulators in the regulation of erythropoiesis after kidney transplantation. Thirty-six kidney graft recipients with stable graft function for more than 12 months were studied. According to the hemoglobin levels they were divided into: group 1 (12 patients) with normal graft function (sCr = 145.2 +/- 15.8 micromol/l) and normal hemoglobin (12.7+/-0.3 g/dl), group 2 (11 patients) with normal graft function (sCr = 135 +/- 6.5 miromol/l) and posttransplant erythrocytosis (Hb = 18.1 +/- 0.2 g/dl) and group 3 (13 patients) with chronic graft failure (sCr = 223.7 +/- 28.9 micromol/l, range 181-294) and anemia (Hb = 9.0 +/- 0.8 g/dl). Early erythroid progenitors (BFU-E) from peripheral blood, serum immunoreactive Epo and burst promoting activity (BPA) in PHA-LCM prepared from patients' peripheral blood mononuclear cells were measured in all studied patients. The expected Epo for Hb was found normal in patients with normal graft function, 10 times higher in patients with PTE and low in patients with anemia. BPA in PHA-LCM prepared from PTE was increased in 4/6 patients, normal in 4/6 anemic patients, but it was decreased in 5 patients with normal Hb. The mean values were 20.8 +/- 6.3 in PTE group and 16.2 +/- 6.8 in anemic group, and 4.1 +/- 1.8 (at the level of normal controls) in group 1. The number of BFU-E derived colonies was low in most patients with normal hemoglobin and anemia, and increased in most patients with PTE. Spontaneous BFU-E colonies i.e. without Epo added to the cultures were found in 7 of 12 patients with PTE. The mean values of BFU-E showed significant differences between patients with PTE (17.43 +/- 7.3), and patients with normal hemoglobin and anemia (4.39 +/- 1.2 vs. 6.5 +/- 1.1). The results presented suggest that inappropriate Epo secretion depends on the graft function and is the primarily important regulator that caused PTE or anemia after kidney transplantation. Synergistic action of BPA with Epo as well as increased sensitivity of early erythroid precursors to these stimulators could explained sustained erythropoiesis in PTE patients. The high BPA levels in anemic transplant patients with moderate chronic graft failure could be beneficial if rHuEpo treatment is applied in this patient group.

In vivo effects of recombinant human erythropoietin on bone marrow hematopoiesis in patients with chronic renal failure.

Studies of hemopoietic progenitors and precursors in bone marrow before and after two months of recombinant human erythropoietin (rhEpo) therapy in 12 patients with uremic anemia are the subject of this investigation caried out in order to have a better insight into the effect of Epo in vivo. Eight patients were on hemodialysis and four others were predialysis patients with chronic renal failure. The starting dose of rhEpo was 30-50 U/kg bw and was increased by 50 percent every four weeks. The mean hemoglobin values rose from 6.08 +/- 1.03 to 9.8 +/- 1.98 g/dl at the time of study. The number of bone marrow derived erythroid colonies, both early (BFU-E) and late (CFU-E) were found to be higher than subnormal values, found before the therapy. The percentage of erythroid progenitors in cell cycle increased to higher than normal values for BFU-E and to normal values for CFU-E. At the same time granulocytic progenitors (CFU-GM) decreased to the range of normal values (67.3 per 10 superset5 cells). Slightly increased Epo levels (approx. 30mU/ml) during the replacement therapy were optimal for correction of anemia. The rhEpo therapy induced an increase of percentage of erythroblasts and the decrease of myeloid to erythroid ratio (M/E) in the bone marrow. Only in predialysis patients in whom the target hemoglobin values were achieved by rhEpo therapy at the time of the study the percentage of erythroblasts in the bone marrow increased to normal values. Increase of erythroblasts in bone marrow in patients under two months of substitutive therapy with rhEpo with the increase of both, early and late erythroid progenitors we have observed, is significant indicating the stimulative effect of rhEpo on all subsets of erythropoiesis leading to normalization of erythropoiesis at all levels. No stimulative effect of rhEpo replacement therapy on granulopoiesis was observed.

Factors inducing posttransplant erythrocytosis.

In this study factors possibly contributing to the development of erythrocytosis after renal transplantation (PTE) were analyzed. Out of 131 transplanted patients nine developed PTE (mean hemoglobin 17. 9 +/- 0.3 g/dl) 2 to 27 months after transplantation (group 1) and were compared to the nine with normal hemoglobin concentration (mean hemoglobin 12.4 +/- 0.2 g/dl, control group 2). The study was performed about two years after transplantation (25 +/- 3.9 months group 1 and 23.7 +/- 2.6 months group 2). Immunosuppressive therapy given in standard doses consisted of cyclosporine, azathioprine and prednisone. At the onset of the study no difference in renal graft function was noted between the groups (for group 1 sCr = 111.7 +/- 10.4 micromol/l and for group 2 sCr = 154.6 +/- 27.6 micromol/l). The mean serum immunoreactive erythropoietin (Epo) levels were significantly higher in PTE patients compared to control group of patients (33.9 +/- 4.6 mU/ml vs 21.6 +/- 2.5 mU/ml, p = 0.03). In addition, the ratio between observed to expected (O/E) Epo, a useful index in assessing Epo secretion in renal transplant patients, was ten times higher for group 1 than for group 2 (Median value 10.0 vs. 1.05). Spontaneous growth of Burst-forming unit- erythroid (BFU-E) in peripheral blood was detected in 5 out of 9 patients from group 1 and none in patients from group 2 (p = 0.04). Burst Promoting Activity (BPA) in Phytohemagglutinine Stimulated Leukocytes Condition Medium (PHA-LCM) from patients blood were higher in the PTE patients than in controls. Whole blood cyclosporine levels were higher in group 1 than in group 2 throughout the first 30 weeks after transplantation. It was concluded that sustained erythropoiesis after correction of renal anemia by kidney transplantation, leading to PTE could be explained as a consequence of increased levels of Epo and BPA and increased sensitivity of early erythroid progenitors to these stimulators induced by high cyclosporine levels.

Erythropoietin & erythroid progenitors in rats exposed to chronic hypoxia.

In order to better understand the mechanisms affecting erythropoietin (Epo) synthesis and red cell mass increase under chronic hypoxia, we examined Epo production and erythroid progenitors (CFU-E) in rats exposed to normobaric hypoxia for four weeks. Hypoxia induced the rise of hematocrit (Htc), hemoglobin (Hb) concentration and the red blood cell (RBC) number with a plateau in hematocrit values after two weeks. After 24 h of hypoxia, Epo levels were increased 20 fold, followed by a significant decrease. After the first week of hypoxia, the values were still higher than in the controls, but after two weeks Epo levels did not differ significantly from the normal values. The fall of Epo levels coincided with the plateau values of hematocrit. The changes in the CFU-E number followed the changes in Epo concentration: a two fold increase after 24 h of hypoxia; a further increase during the next two weeks reaching a peak on day 14, and then a progressive decrease at the time when Epo concentration was at a normal level. Although decreased, but still higher than normal, the CFU-E number during the last two weeks of hypoxia could be necessary for the maintenance of an achieved steady state under persistent hypoxic conditions with normal Epo concentration sufficient to maintain the existing rate of erythropoiesis.

Recombinant human erythropoietin treatment of anemia in renal transplant patients.

The rHuEpo effect on anemia in eight renal transplant patients (group A) with severe anemia (Hb 6.0-7.5 g/dL) and chronic graft failure (CGF) (sCr 281-794 mumol/L) was compared to the rHuEpo effect on anemia in predialysis (20 patients-group B) and hemodialysis patients (17 patients-group C) in order to examine the rHuEpo effect on anemia and graft failure progression, and to find out whether the response to therapy in these three patient groups differed. Although renal function impairment was similar in patients from group A and B, anemia was more severe in patients from group A. Serum immunoreactive erythropoietin levels were within normal limits for nonanemic persons, that is, inadequate for the level of anemia in all patients before therapy. Maintenance immunosuppression given after renal transplantation consisted of cyclosporine, azathioprine, and prednisone in standard doses. The startig rHuEpo dose of 150 U/kg/wk increased by 25 U/kg if the target Hb of 10.0 g/dL was not achieved at the end of a 4-week period. When target Hb was achieved, the rHuEpo dose was regularly adjusted to maintain Hb of 10.0 g/dL. Most patients from group A and group C were polytransfused before rHuEpo therapy and consequently with iron overload so that only some patients from these groups and all predialysis patients needed iron supplementation given orally. Anemia improved in all patients with 2 to 10 weeks of treatment. Mean rHuEpo doses for the first 2 months were similar in three studied groups, but the patients with the lowest initial hemoglobin values responded better to rHuEpo therapy. The rate of Hb increase during the initial phase of therapy was significantly higher in patients from group A and B comparing to patients from group C, indicating the importance of residual renal function for rHuEpo effect on anemia. Progression of CGF expressed by the slope of l/sCr vs. time did not change in either patients from group A or in predialysis patients. It could be concluded that rHuEpo therapy improved anemia in transplant patients as in predialysis and hemodialysis patients. Anemia improvement by rHuEpo did not accelerate the progression of graft function.

Hematopoietic growth factors in anemia of Belgrade laboratory (b/b) rats.

In this study, the extent to which growth factor production and microenvironment might be responsible for defective erythropoiesis and granulopoiesis in anemic b/b rats is investigated. Radioimmunoassay-determined serum erythropoietin (Epo) levels are high in b/b rats and closely related to degree of anemia. The low number of erythroid progenitors in b/b rats despite a high Epo level suggested that the defective erythropoiesis could be due to a low level of burst-promoting activity (BPA). A pokeweed mitogen-stimulated medium (PWM-SCM) was prepared with b/b rat spleen cells and used in normal and anemic rat bone marrow and spleen cultures to determine BPA and other growth factor levels. No erythroid burst-forming unit-derived colonies were found but granulocyte-macrophage colony-forming units were counted in significant number, suggesting that the production of growth factors that supports the growth of granulopoietic progenitors is not significantly disturbed. Because BPA is produced mainly by T-lymphocytes, the low BPA level in b/b rat PWM-SCM raised the question of the functional capacity of T-lymphocytes. Investigations showed a decrease in the proliferative activity of b/b rat spleen mitogen-activated T-lymphocytes to about 20% of controls as well as a decrease in interleukin-2 activity in b/b rat spleen cell supernatants. These results point to defective T-lymphocytes. A study of bone marrow fibroblastoid cell colonies (CFU-F) revealed significantly lower CFU-F counts in the b/b rats. This finding is indicative of a disturbed microenvironment, which could also to some extent be responsible for decreased growth factor production and depressed hematopoiesis in the b/b rat.

[Regulation of erythropoiesis by erythropoietin]. / Regulacija eritropoeze eritropoetinom.

Regulation of erythropoiesis by erythropoietin, proposed already at the beginning of this century, has been greatly enhanced by cloning and expression of the gene of erythropoietin. Erythropoietin gene functions as a housekeeping gene, so that erythropoietin is constitutively produced and is never absent from the blood. Hypoxia activates erythropoietin gene primarily in the kidney and also to a small extent in the liver. Peritubular interstitial cells in the kidney or the kidney tubular cells produce erythropoietin. The modulation of erythropoietin gene expression in the kidney and the liver differs. Kidney cells produce erythropoietin in all-or-non pattern and as the stimulus becomes more severe more cells produce erythropoietin while in the liver individual cells produce more erythropoietin when the stimulus is more severe. Erythropoietin exerts its effect on erythroid progenitor cells after binding to specific receptors that are expressed on target cells, interacts with those cells in the bone marrow stimulating them into cell cycle, supporting the survival of cycling progenitor cells and permitting their final differentiation into cells synthetizing hemoglobin.

Serum erythropoietin levels in hemodialysed patients after administration of recombinant human erythropoietin.

In anemic patients on regular hemodialysis (HD), correction of anemia with recombinant human erythropoietin (rHuEpo) administered intravenously (iv) or subcutaneously (sc) was followed over a 2-month period. Monitoring serum Epo post-dose concentrations after the first iv rHuEpo injection and following another regular injection after 2 months of therapy with rHuEpo iv in 9 patients showed that the Epo elimination half-life was reduced from 7.48 h to 4.68 h. In the same patients the initially low percentage of erythroblasts and mature erythroid progenitors increased during 2 months of rHuEpo therapy. Because Epo molecules bound to Epo receptors are internalized in target cells we suggest that the expansion of the Epo responsive cell pool could explain the shorted Epo elimination time after 2 months of rHuEpo treatment. By monitoring serum Epo concentration following sc rHuEpo injection in 7 HD patients it was found that the modest increase in serum Epo levels (30-60 mU/ml) was sufficient to correct anemia.

Anemia in Balkan endemic nephropathy.

The severity of anemia in patients at different stages of the evolution of two tubulointerstitial nephropathies, Balkan endemic nephropathy and chronic pyelonephritis, was compared to clarify the previous observations that anemia appears earlier and is more severe in Balkan endemic nephropathy than in other renal diseases. The role of erythropoietin insufficiency as the cause of anemia in endemic nephropathy was studied as well. The severity of anemia increased with the impairment of renal function in endemic nephropathy and was similar to anemia in chronic pyelonephritis. However, in patients with endemic nephropathy at the initial stage of renal insufficiency significantly lower red cell concentrations were found compared with control subjects from the endemic region. In contrast, patients with pyelonephritis did not have decreased red cell concentrations at the early phase of their renal failure, suggesting that earlier appearance of anemia is characteristic for endemic nephropathy. To confirm this finding a study involving larger number of patients would be necessary. The serum erythropoietin levels, inappropriately low for the degree of anemia in patients with renal failure, were unrelated to the type of tubulointerstitial nephropathy.

Abnormal megakaryocytopoiesis in the Belgrade laboratory rat.

The Belgrade laboratory (b/b) rat has a hereditary hypochromic microcytic anemia because of defective transmembrane iron transport into erythroblasts. The present study was prompted by our previous work in which we showed that the b/b rat has hypomegakaryocytic thrombocytopenia associated with increased megakaryocyte size. To define the basic mechanism underlying this abnormality in the b/b rat we have studied both megakaryocytopoiesis and granulopoiesis in anemic b/b rats, chronically transfused b/b rats, iron-treated b/b rats, and controls. We have found decreased concentrations of megakaryocyte and granulocyte progenitors in the marrow of b/b rats. Full correction of the severe anemia by chronic transfusion resulted in normalization of megakaryocyte progenitors, small acetylcholinesterase positive cells, megakaryocyte size, and platelet counts, along with granulocyte progenitors. In contrast, the partial correction of anemia obtained by iron treatment resulted in improvement, but not normalization, of these parameters. These findings indicate that abnormal megakaryocytopoiesis in the b/b rat can be best interpreted as a consequence of hypoxia because of the severe anemia. Because we have recently shown that the number of erythroid progenitors in b/b rats is also low, we propose that abnormal megakaryocytopoiesis in this animal is a reflection of an acquired stem cell disorder induced by the prolonged hypoxia resulting from the severe anemia.

Erythropoietin and improvement of anemia in long-term hemodialysis patients.

Two groups, with 4 patients each were selected for study out of 155 patients on regular hemodialysis (HD): Group I, with hematocrit (PCV) less than 20% and group II, with PCV greater than 30%. The patients in both groups had been anemic at the start of HD treatment, but a significant improvement in their anemia had occurred only among the patients in the Group II. The main difference between the two patient groups, other than the degree of anemia, was found to be in serum erythropoietin (Ep) levels. No significant differences were observed between the two groups in serum urea, creatinine, parathyroid hormone or CFU-E growth inhibition. Acquired cystic disease of the kidney was found in five patients from group I, and in 11 patients from group II. The correlation between the number of cysts in the kidneys and the patient's PCV and serum Ep levels proved significantly positive. The results presented could be regarded as another proof that diseased kidney is capable of functioning as an Ep producing organ despite the loss of excretory function.

Endogenous BFU-E in peripheral blood in diagnosis of polycythemia vera.

Erythroid progenitors (CFU-E and BFU-E) growth in vitro from bone marrow and peripheral blood of patients with polycythemia vera (PV) was studied using a methylcellulose culture technique. The aim of the study was to find out whether the in vitro colony formation of peripheral blood could be used in the differential diagnosis of PV. In all 25 patients studied, endogenous colonies were found in the bone marrow and peripheral blood. The parallel study of both bone marrow and peripheral blood erythroid progenitors indicates that the presence of endogenous BFU-E in peripheral blood is a dependable test for PV. The results presented here showed that the abnormalities in PV erythroid progenitors are expressed at the level of both CFU-E and BFU-E, suggesting multiple changes in the erythroid progenitors. Our finding indicate that peripheral blood BFU-E differ from bone marrow BFU-E with regard to their dependence for further differentiation on BPA, the activity present in PHA-LCM.

Granulopoiesis in anemic Belgrade laboratory (b/b) rats.

The aim of this study was to clarify the nature of the abnormalities of granulopoiesis in anemic b/b rats. The size of different granulocytic cell compartments (granulocytic and macrophage precursor cells, proliferative and nonproliferative morphologically recognizable granulocytic cells) in the bone marrow, spleen, and peripheral blood was determined, and activity of granulocytic stimulators (colony-stimulating activities derived from lung-conditioned medium of endotoxin-treated rats and from spleen cells stimulated in vitro with pokeweed mitogen) was investigated. Depressed bone marrow granulopoiesis and expanded granulopoiesis in the spleen, together with the sustained production of granulocytic stimulators, was found in anemic b/b rats. It is suggested that the changes within the bone marrow microenvironment are the essential cause of disturbances of granulopoiesis in anemic b/b rats. The increased proliferation of granulocytic cells in iron-treated b/b rats indicates a role of iron in the cellular proliferation.

Megakaryocytopoiesis in experimentally induced chronic normobaric hypoxia.

It was recently proposed that prolonged hypoxia produces hypomegakaryocytic thrombocytopenia by reducing the pool of committed megakaryocyte progenitor cells at the expense of a greatly expanded erythroid progenitor pool. In order to test this hypothesis we have studied the relationship between megakaryocytopoiesis, erythropoiesis, and granulopoiesis at the level of progenitor cells (megakaryocyte colony-forming unit, CFU-Mk; erythroid CFU, CFU-E; erythroid burst-forming units; BFU-E; and granulocyte-macrophage CFU, CFU-GM) in the marrow of rats exposed for 4 weeks to normobaric hypoxia. We have found that hypomegakaryocytic thrombocytopenia was accompanied by decreased CFU-Mk, increased CFU-E, and a normal number of BFU-E and CFU-GM. These results support the hypothesis that prolonged hypoxia reduces the precursor cell commitment to differentiate into the megakaryocyte series by enhancing demand for differentiation into the erythroid cell line. However, the underlying mechanism needs further investigation.

Erythropoietin and anemia in the progression of Balkan endemic nephropathy and other renal diseases.

We have investigated anemia in patients at different stages of the evolution of three chronic renal diseases: Balkan endemic nephropathy (BEN), chronic pyelonephritis (PN) and chronic glomerulonephritis (GN). A total of 88 patients with creatinine clearances from 9 to 118 ml/min and hemoglobin concentrations from 70 to 160 g/l were studied with regard to the relationship, if any, between erythropoietin production and the type and stage of nephropathy. Anemia in BEN was a particular focus of interest since it had been stated that in BEN, anemia precedes renal failure. Our data neither prove nor disprove this statement. A significant positive correlation between creatinine clearance and hemoglobin concentration was found in all three nephropathies, indicating that in the patients studied the severity of anemia increased with the impairment of renal function regardless of the underlying disease. Serum levels of immunoreactive erythropoietin were in the normal range in 54 patients, moderately increased in 20 and slightly decreased in 14. The erythropoietin level appears to be unrelated to the stage of renal failure or the type of nephropathy. The only exception was the subgroup where the patients with glomerulonephritis and normal renal function had increased serum erythropoietin levels and significantly higher parameters of red blood cell concentration than the patients from the same subgroup with tubulointerstitial nephropathies. In patients with severe renal failure and anemia, serum erythropoietin levels were inappropriately low for the degree of anemia, indicating that erythropoietin plays a role in the pathogenesis of the anemia.

Erythropoiesis in paroxysmal nocturnal hemoglobinuria.

Ten patients with paroxysmal nocturnal hemoglobinuria were studied. The diagnosis was made on the basis of hemolytic anemia, a positive Ham test and hemosiderinuria. Six patients had primary paroxysmal nocturnal hemoglobinuria evolved from aplastic anemia. These four patients also had a milder form of the disease, Over long periods of the follow-up, large variations of hemoglobin values and red blood cell counts were observed. Both absolute and percent reticulocyte counts were increased. Erythroblast counts in the bone marrow were 3-5 times higher than normal. Reticulocyte counts in the bone marrow were 3-5 times higher than normal. Reticulocyte counts showed wide variations but substantially smaller than those in autoimmune hemolytic anemias. Serum iron was either normal or increased, while the bone marrow iron store was high or low. However, the finding of urinary hemosiderin in all cases spoke against depletion of iron stores. The red blood cell life span was moderately shortened. Kinetic studies with 59Fe showed a high red blood cell iron incorporation, while the curves frequently had irregular shapes (broken curve) or an early, abrupt fall. Studies of late erythroid progenitors (CFU-E) indicated that this compartment was preserved. Even after long observation periods was no stem cell pool depletion due to an increased red blood cell demand observed.

[Erythropoietin]. / Eritropoetin.

New data on erythropoietin and regulation of erythropoiesis are presented; the possibility to treat anaemia in patients with chronic renal disease by erythropoietin is stressed.

Erythroid progenitors in anemic Belgrade laboratory (b/b) rats.

The anemia of Belgrade b/b rats has been shown to be due to intracellular iron deficiency. The aim of this study of erythropoiesis at the progenitor cell level in these rats was to determine if a defect is present in the early phase of red cell production. Both erythroid colony-forming unit (CFU-E)- and erythroid burst-forming unit (BFU-E)-derived colonies were found to be few in untreated b/b rats and made up of a small number of poorly hemoglobinized erythroblasts of different size and irregular cell shape. Following treatment with iron, the anemia of the rats improved, and the number of CFU-E-derived colonies and the number of cells per colony increased, but the peculiar erythroblast morphology persisted. The high serum level of biologically active erythropoietin (Ep) in b/b rats rules out inadequate Ep production as a cause of their anemia. The results presented indicate, in addition to the earlier described defective transmembrane iron transport, a defect in erythroid progenitor cells. The effect of iron treatment in these rats detected in vitro on erythroid progenitors confirms the importance of iron for cellular proliferation.