17Beta-estradiol decreases hypoxic induction of erythropoietin gene expression

Exposure to chronic hypoxia induces erythropoietin (EPO) production to facilitate oxygen delivery to hypoxic tissues. Previous studies from our laboratory found that ovariectomy (OVX) exacerbates the polycythemic response to hypoxia and treatment with 17beta-estradiol (E2-beta) inhibits this effect. We hypothesized that E2-beta decreases EPO gene expression during hypoxia. Because E2-beta can induce nitric oxide (NO) production and NO can attenuate EPO synthesis, we further hypothesized that E2-beta inhibition of EPO gene expression is mediated by NO. These hypotheses were tested in OVX catheterized rats treated with E2-beta (20 microg/day) or vehicle for 14 days and exposed to 8 or 12 h of hypoxia (12% O(2)) or normoxia. We found that E2-beta treatment significantly decreased EPO synthesis and gene expression during hypoxia. E2-beta treatment did not induce endothelial NO synthase (eNOS) expression in the kidney but potentiated hypoxia-induced increases in plasma nitrates. We conclude that E2-beta decreases hypoxic induction of EPO. However, this effect does not appear to be related to changes in renal eNOS expression.     

The effects of interferon on murine erythropoiesis

The effects of interferon (IF) on erythropoietin (Ep) action and production were studied in mice. In comparison to control animals, Ep action in exhypoxic, polycythemic mice was significantly decreased (p less than 0.05) following two low dose injections of IF (2.9-3.5 X 10(4) units). In addition, renal Ep production in normal intact mice was also significantly decreased (p less than 0.01) following a single injection of IF (5.4-6.3 X 10(4) units) and hypoxic exposure.     

Time-course of the polycythemic response in normoxic and hypoxic mice with high blood oxygen affinity induced by cyanate administration

The Hb-O₂ affinity and the erythropoietic response as a function of time were studied in mice treated with sodium cyanate for up to 2 months. Cyanate increased the Hb-O₂ affinity in normoxic mice more than in chronically hypoxic mice. The hemoglobin concentration rose as a function of time both in normoxic and hypoxic conditions but reached higher levels in hypoxia. After 42 days of study (21 days of hypoxia) hemoglobin reached maximum levels and thereafter showed a plateau in both cyanate and control animals. It is concluded that a chronic left-shifted oxygen dissociation curve does not avoid the development of hypoxic polycythemia in mice. Moreover, prolonged cyanate administration potentiates the crythropoietic response to chronic hypoxia. Since polycythemia is an index of tissue hypoxia, the results show that the high hemoglobin affinity did not prevent tissue hypoxia in low PO₂ conditions. Results showing beneficial effects of high hemoglobin oxygen affinity induced by cyanate based on acute hypoxic expositions should be cautiously interpreted with regard to their adaptive value in animals chronically exposed to natural or simulated hypoxia.Abbreviations Hb hemoglobin - NaOCN sodium cyanate - ODC oxygen dissociation curve - P ₅₀ PO₂ at which hemoglobin is half saturated with O₂     

The negative regulation of red cell mass by neocytolysis: physiologic and pathophysiologic manifestations.

We have uncovered a physiologic process which negatively regulates the red cell mass by selectively hemolyzing young circulating red blood cells. This allows fine control of the number of circulating red blood cells under steady-state conditions and relatively rapid adaptation to new environments. Neocytolysis is initiated by a fall in erythropoietin levels, so this hormone remains the major regulator of red cell mass both with anemia and with red cell excess. Physiologic situations in which there is increased neocytolysis include the emergence of newborns from the hypoxic uterine environment and the descent of polycythemic high-altitude dwellers to sea level. The process first became apparent while investigating the mechanism of the anemia that invariably occurs after spaceflight. Astronauts experience acute central plethora on entering microgravity resulting in erythropoietin suppression and neocytolysis, but the reduced blood volume and red cell mass become suddenly maladaptive on re-entry to earth's gravity. The pathologic erythropoietin deficiency of renal disease precipitates neocytolysis, which explains the prolongation of red cell survival consistently resulting from erythropoietin therapy and points to optimally efficient erythropoietin dosing schedules. Implications should extend to a number of other physiologic and pathologic situations including polycythemias, hemolytic anemias, 'blood-doping' by elite athletes, and oxygen therapy. It is likely that erythropoietin influences endothelial cells which in turn signal reticuloendothelial phagocytes to destroy or permit the survival of young red cells marked by surface molecules. Ongoing studies to identify the molecular targets and cytokine intermediaries should facilitate detection, dissection and eventual therapeutic manipulation of the process.     

Mimicking Hypoxia to Treat Anemia: HIF-Stabilizer BAY 85-3934 (Molidustat) Stimulates Erythropoietin Production without Hypertensive Effects

Oxygen sensing by hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) is the dominant regulatory mechanism of erythropoietin (EPO) expression. In chronic kidney disease (CKD), impaired EPO expression causes anemia, which can be treated by supplementation with recombinant human EPO (rhEPO). However, treatment can result in rhEPO levels greatly exceeding the normal physiological range for endogenous EPO, and there is evidence that this contributes to hypertension in patients with CKD. Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production. HIF stabilization by oral administration of the HIF-PH inhibitor BAY 85-3934 (molidustat) resulted in dose-dependent production of EPO in healthy Wistar rats and cynomolgus monkeys. In repeat oral dosing of BAY 85-3934, hemoglobin levels were increased compared with animals that received vehicle, while endogenous EPO remained within the normal physiological range. BAY 85-3934 therapy was also effective in the treatment of renal anemia in rats with impaired kidney function and, unlike treatment with rhEPO, resulted in normalization of hypertensive blood pressure in a rat model of CKD. Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin–angiotensin system. Thus, BAY 85-3934 may provide an approach to the treatment of anemia in patients with CKD, without the increased risk of adverse cardiovascular effects seen for patients treated with rhEPO. Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia.     

Chronic anemia and effects of iron supplementation in a research colony of adult Rhesus macaques (Macaca mulatta)

A cohort of rhesus macaques used in neuroscience research was found at routine examinations to have chronic anemia (spun Hct less than 30%). Four anemic (Hct, 24.8% ± 3.4%) and 10 control (39.6% ± 2.9%) macaques were assessed to characterize the anemia and determine probable cause(s); some animals in both groups had cephalic implants. Diagnostic tests included CBC, bone marrow evaluations, iron panels, and serum erythropoietin and hepcidin concentrations. Serum iron and ferritin were 15.8 ± 11.1 μg/dL and 103.8 ± 53.1 ng/mL, respectively, for the anemic group compared with 109.8 ± 23.8 μL/dL and 88.5 ± 41.9 ng/mL, respectively, for the control group. Erythropoietin levels were 16.2 to over 100 mU/mL for the anemic macaques compared with 0 to 1.3 mU/mL for the control group. Hepcidin results were similar in both groups. Because the findings of low iron, high erythropoietin, and normal hepcidin in the anemic macaques supported iron-deficiency anemia or anemia of chronic disease combined with iron-deficiency anemia, a regimen of 4 doses of iron dextran was provided. In treated macaques, Hct rose to 36.3% ± 6.8%, serum iron levels increased to 94.0 ± 41.9 μg/dL, and erythropoietin levels fell to 0.15 to 0.55 mU/mL. Maintenance of normal Hct was variable between macaques and reflected individual ongoing clinical events.     

Role of the spleen in the exaggerated polycythemic response to hypoxia in chronic mountain sickness in rats.

In a rat model of chronic mountain sickness, the excessive polycythemic response to hypoxic exposure is associated with profound splenic erythropoiesis. We studied the uptake and distribution of radioactive iron and red blood cell (RBC) morphology in intact and splenectomized rats over a 30-day hypoxic exposure. Retention of (59)Fe in the plasma was correlated with (59)Fe uptake by both spleen and marrow and the appearance of (59)Fe-labeled RBCs in the blood. (59)Fe uptake in both the spleen and the marrow paralleled the production of nucleated RBCs. Splenic (59)Fe uptake was approximately 10% of the total marrow uptake under normoxic conditions but increased to 60% of the total marrow uptake during hypoxic exposure. Peak splenic (59)Fe uptake and splenomegaly occurred at the most intense phase of erythropoiesis and coincided with the rapid appearance of (59)Fe-labeled RBCs in the blood. The bone marrow remains the most important erythropoietic organ under both resting and stimulated states, but inordinate splenic erythropoiesis in this rat strain accounts in large measure for the excessive polycythemia during the development of chronic mountain sickness in chronic hypoxia.     

POLYCYTHEMIA VERA—Peripheral Vascular Manifestations

The presenting manifestations of polycythemia vera are often complications involving the vascular system. These include myocardial infarction, cerebro-vascular accidents and ischemic changes in the extremities.The concept of increased atherogenesis in cases of polycythemia vera has been questioned. A possible mechanism by which small, otherwise subclinical atheromatous plaques produce ischemic symptoms in patients with polycythemia vera is discussed. The blood in polycythemic patients has been shown to have an increased viscosity resulting in a prolonged circulation time. If a small atheromatous plaque is present in association with increased blood viscosity, this combination may well produce ischemic symptoms. This explains why treatment of polycythemia vera, with restoration of blood to normal viscosity, often reverses the patient''s ischemic symptoms.Two cases of polycythemia vera here reported, in which the presenting manifestations were gangrenous extremities, emphasize the need for prompt diagnosis and treatment of polycythemia vera. In the first case, early recognition and treatment of polycythemia vera successfully reversed the ischemic changes in the extremities, while failure of early recognition and treatment in the second case resulted in two major amputations.     

Effects of polycythemia on systemic endothelial function in chronic hypoxic lung disease

Chronic obstructive pulmonary disease (COPD) is a major risk factor for cardiovascular disease. Polycythemia, a common complication of hypoxic COPD, may affect systemic vascular function by altering blood viscosity, vessel wall shear stress (WSS), and endothelium-derived nitric oxide (NO) release. Here, we evaluated the effects of hypoxia-related polycythemia on systemic endothelial function in patients with COPD. We investigated blood viscosity, WSS, and endothelial function in 15 polycythemic and 13 normocythemic patients with COPD of equal severity, by recording brachial artery diameter variations in response to hyperemia and by using venous occlusion plethysmography (VOP) to measure forearm blood flow (FBF) responses to a brachial artery infusion of acetylcholine (ACh), bradykinin (BK), sodium nitroprusside (SNP), substance P (SP), isoptin, and N-monomethyl-L-arginine (L-NMMA). At baseline, polycythemic patients had higher blood viscosity and larger brachial artery diameter than normocythemic patients but similar calculated WSS. Flow-mediated brachial artery vasodilation was increased in the polycythemic patients, in proportion to the hemoglobin levels. ACh-induced vasodilation was markedly impaired in the polycythemic patients and negatively correlated with hemoglobin levels. FBF responses to endothelium- (BK, SP) and non-endothelium-dependent (SNP, isoptin) vasodilators were not significantly different between the two groups. L-NMMA infusion induced a similar vasoconstrictor response in both groups, in accordance with their similar baseline WSS. In conclusion, systemic arteries in polycythemic patients adjust appropriately to chronic or acute WSS elevations by appropriate basal and stimulated NO release. Overall, our results suggest that moderate polycythemia has no adverse effect on vascular function in COPD.     

Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE2 and in specified samples also of 6-keto-PGF1 alpha, as a measure of PGI2, and PGF2 alpha were determined by radioimmunoassay after incubation at either 20% O2 (normoxic) or 2% O2 (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE2 production was noted among independent cell lines. PGE2 production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE2 production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF2 alpha and 6-keto-PGF1 alpha increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.     

Fetal anemia leads to augmented contractile response to hypoxic stress in adulthood

In response to chronic fetal anemia, coronary blood flow, maximal coronary conductance, and coronary reserve increase. We sought to determine whether chronic fetal anemia alters left ventricular (LV) function in adulthood. We studied adult sheep that had been made anemic for 20 days in utero by phlebotomy. They were transfused just before birth. At 7 mo of age, LV function was measured by pressure-volume loops at rest and during hypoxic stress. The in utero anemia group (n = 8) did not differ from controls (n = 5) with respect to hematocrit, heart and body weight, or baseline hemodynamic parameters. However, the effect of hypoxia (relative to baseline) on multiple indexes of systolic function was different between the two groups. End-systolic elastance increased in the in utero anemia group (baseline to hypoxia) by 4.15 +/- 3.47 mmHg/ml (mean +/- SD) but changed little in controls (0.24 +/- 0.45), which shows that the response to hypoxia was significantly different (P < 0.01) between groups. Similarly, the maximum derivative of LV pressure with respect to time increased in the in utero anemia group (486 +/- 340 mmHg/s,) but on average fell in the controls (-503 +/- 211 mmHg/s) with the response again being significantly different (P < 0.03). We conclude that in sheep, perinatal anemia can alter cardiac responses to hypoxic stress in the adult long after restoration of normocythemia.     

Regulation of canine skeletal muscle and hindlimb blood flow in acute anemia

Redistribution of blood flow away from resting skeletal muscles does not occur during anemic hypoxia even when whole body oxygen uptake is not maintained. In the present study, the effects of sympathetic nerve stimulation on both skeletal muscle and hindlimb blood flow were studied prior to and during anemia in anesthetized, paralyzed, and ventilated dogs. In one series (skeletal muscle group, n = 8) paw blood flow was excluded by placing a tourniquet around the ankle; in a second series (hindlimb group, n = 8) no tourniquet was placed at the ankle. The distal end of the transected left sciatic nerve was stimulated to produce a maximal vasoconstrictor response for 4-min intervals at normal hematocrit (Hct.) and at 30 min of anemia (Hct. = 14%). Arterial blood pressure and hindlimb or muscle blood flow were measured; resistance and vascular hindrance were calculated. Nerve stimulation decreased blood flow (p less than 0.05) in the hindlimb and muscle groups at normal Hct. Blood flow rose (p less than 0.05) during anemia and was decreased (p less than 0.05) in both groups during nerve stimulation. However, the blood flow values in both groups during nerve stimulation in anemic animals were greater (p less than 0.05) than those at normal Hct. Hindlimb and muscle vascular resistance fell significantly during anemia and nerve stimulation produced a greater increase in vascular resistance at normal Hct. Vascular hindrance in muscle, but not hindlimb, was less during nerve stimulation in anemia than at normal Hct.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of adrenomedullin in hypoxic and ischemic rat kidneys and human kidneys with arterial stenosis

To investigate regional aspects of hypoxic regulation of adrenomedullin (AM) in kidneys, we mapped the distribution of AM in the rat kidney after hypoxia (normobaric hypoxic hypoxia, carbon monoxide, and CoCl(2) for 6 h), anemia (hematocrit lowered by bleeding) and after global transient ischemia for 1 h (unilateral renal artery occlusion and reperfusion for 6 and 24 h) and segmental infarct (6 and 24 h). AM expression and localization was determined in normal human kidneys and in kidneys with arterial stenosis. Hypoxia stimulated AM mRNA expression significantly in rat inner medulla (CO 13 times, 8% O(2) 6 times, and CoCl(2) 8 times), followed by the outer medulla and cortex. AM mRNA level was significantly elevated in response to anemia and occlusion-reperfusion. Immunoreactive AM was associated with the thin limbs of Henle's loop, distal convoluted tubule, collecting ducts, papilla surface epithelium, and urothelium. AM labeling was prominent in the inner medulla after CO and in the outer medulla after occlusion-reperfusion. The infarct border zone was strongly labeled for AM. In cultured inner medullary collecting duct cells, AM mRNA was significantly increased by hypoxia. AM mRNA was equally distributed in human kidney and AM was localized as in the rat kidney. In human kidneys with artery stenosis, AM mRNA was not significantly enhanced compared with controls, but AM immunoreactivity was observed in tubules, vessels, and glomerular cells. In summary, AM expression was increased in the rat kidney in response to hypoxic and ischemic hypoxia in keeping with oxygen gradients. AM was widely distributed in the human kidney with arterial stenosis. AM may play a significant role to counteract hypoxia in the kidney.     

Hemodilutional anemia is associated with increased cerebral neuronal nitric oxide synthase gene expression.

Severe hemodilutional anemia may reduce cerebral oxygen delivery, resulting in cerebral tissue hypoxia. Increased nitric oxide synthase (NOS) expression has been identified following cerebral hypoxia and may contribute to the compensatory increase in cerebral blood flow (CBF) observed after hypoxia and anemia. However, changes in cerebral NOS gene expression have not been reported after acute anemia. This study tests the hypothesis that acute hemodilutional anemia causes cerebral tissue hypoxia, triggering changes in cerebral NOS gene expression. Anesthetized rats underwent hemodilution when 30 ml/kg of blood were exchanged with pentastarch, resulting in a final hemoglobin concentration of 51.0 +/- 1.2 g/l (n = 7 rats). Caudate tissue oxygen tension (Pbr(O(2))) decreased transiently from 17.3 +/- 4.1 to 14.4 +/- 4.1 Torr (P < 0.05), before returning to baseline after approximately 20 min. An increase in CBF may have contributed to restoring Pbr(O(2)) by improving cerebral tissue oxygen delivery. An increase in neuronal NOS (nNOS) mRNA was detected by RT-PCR in the cerebral cortex of anemic rats after 3 h (P < 0.05, n = 5). A similar response was observed after exposure to hypoxia. By contrast, no increases in mRNA for endothelial NOS or interleukin-1beta were observed after anemia or hypoxia. Hemodilutional anemia caused an acute reduction in Pbr(O(2)) and an increase in cerebral cortical nNOS mRNA, supporting a role for nNOS in the physiological response to acute anemia.     

A1 and A2 adenosine receptor regulation of erythropoietin production

The effects of adenosine (ADE) and ADE agonists on erythropoietin (Ep) production were determined using percent (%) 59Fe incorporation in red cells of exhypoxic polycythemic mice. The hemisulfate salt of ADE produced a significant increase in % 59Fe incorporation in response to hypoxia in concentrations of 400 to 1600 nmol/kg/day (i.v.). 5'-N-ethyl-carboxamideadenosine (NECA), a selective A2 receptor agonist, increased radioiron incorporation in a dose-dependent manner (10-100 nmol/kg/day, i.v.). In contrast, N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, did not affect radioiron incorporation in concentrations up to 1600 nmol/kg/day (i.v.). Albuterol, a beta 2-adrenergic agonist, enhanced % 59Fe incorporation in polycythemic mice and low doses of CHA (50 and 100 nmol/kg/day), which were not effective alone on % 59Fe incorporation in polycythemic mice exposed to hypoxia, inhibited the enhancement in radioiron induced by albuterol (25 and 100 micrograms/kg/day, i.p.) plus hypoxia. Theophylline (20 and 80 mg/kg/day, i.p.), a well-known antagonist of ADE receptors, blocked the ADE and NECA enhancement in radioiron incorporation at a dose of theophylline alone which produced only a slight enhancement of % 59Fe incorporation. These results suggest that ADE may both inhibit through A1 receptor activation and increase via A2 receptor stimulation the production of Ep.     

Effects of prostaglandins on erythropoiesis

A model has been presented for the role of the kidney in the physiologic and pathophysiologic control of erythropoiesis. It is postulated that an oxygen deficit created by anemia or hypobaric hypoxia results in the release of prostacyclin and its metabolite 6-keto PGE1, and the release of PGE2 with ischemic hypoxia. Prostacyclin, 6-keto-PGE1, or PGE2 activation of adenylate cyclase, an increase in cyclic AMP, activation of a protein kinase and the phosphorylation of hydrolases, which have been released from lysosomes by hypoxia, lead to increased biosynthesis of erythropoietin (Ep). The mechanism of labilization of lysosomes and the release of hydrolases from these cell organelles is postulated to be related to increases in cyclic GMP levels in a renal cell. An Ep-producing human renal carcinoma cell line grown in tissue culture has been demonstrated to produce significant amounts of PGE2. Meclofenamate, an inhibitor of prostaglandins synthesis, was found to inhibit in vitro production of PGE2, Ep, and dome formation in these renal carcinoma cells, giving support to our hypothesis that pathophysiologic production of Ep tumor cells depends upon prostaglandins production. An Ep-producing clone from this renal carcinoma cell line has been developed that contains low electron density (LED) cells after the cells reach confluency, which show a cytoplasm, with abundant and widely dilated endoplasmic reticulum, an oval nucleus, dispersed chromatin, and prominent nucleoli. These are the cells responsible for dome formation and Ep production. Non-EP-producing clones have also been produced from this renal carcinoma cell line, which did not produce domes even at high cell density and had a distinctly different cell type than the Ep-producing clone. Thus, it is postulated that prostacyclin (PGI2) and its metabolite 6-keto PGE1 play a significant role in hypoxic hypoxia stimulation of Ep production and PGE2 is involved in ischemic hypoxia and renal carcinoma cell production of Ep. A modulating effect of PGE2 and PGD2, the two primary bone marrow prostaglandins, has been proposed in Ep stimulation of the erythroid progenitor cell compartment (CFU-E and BFU-E).