Biomedical subjects
W Fried
Publications and source records attributed to W Fried.
Factors that regulate extrarenal erythropoietin production.
Extrarenal sites are the major source of erythropoietin (Ep) production in fetuses and neonates. Afterwards, however, the kidneys are the major site. The most likely site of Ep production is the liver, although attempts to extract Ep from the liver have been, to date, unsuccessful. Extrarenal Ep production, as is the case for renal Ep production, is regulated primarily by the oxygen demand: supply. However, some differences have been identified in the response of extrarenal and renal sites of Ep production to several conditions. Two classes of conditions that selectively influence extrarenal Ep production are the subject of this paper. These are the following: 1) factors that cause reparable hepatic injury--including partial hepatectomy, CC14, and bile duct ligation, and 2) continuous perfusion of angiotensin II. The differences between the mechanism by which these two factors influence extrarenal Ep production will be explored and discussed.
Regulation of the plasma erythropoietin level in hypoxic rats.
Exposure to hypoxia results in an increase in the plasma erythropoietin (Ep) content to a peak level in 10-12 h. If hypoxia is discontinued before maximum plasma Ep concentrations are reached, then the plasma Ep level continues to rise before it declines toward normal. To clarify this phenomenon, we determined the Ep level in the plasma and kidneys at various times after rats that had been exposed to 0.42 atm returned to ambient pressure. The plasma Ep level 2 h after hypoxia was 4-5 times as high as it was immediately after hypoxia and then gradually declined. The Ep content of the kidneys rose for only 1 h after which it rapidly fell to an undetectable level. The most plausible interpretation of this data is that renal Ep production decelerates rapidly within 1 h after return to ambient pressure, but plasma Ep levels continue to rise because of the release of preformed Ep into the plasma. Experiments were also performed to determine whether an increased plasma Ep level inhibits the production or secretion of Ep. Three different types of experiments were performed in which the plasma Ep level was increased by intravenously injecting a large amount of Ep prior to, during, or after exposure to hypoxia and determining its effect on the plasma Ep activity from 15 min to 4 h after injection. In all experiments the plasma and renal Ep levels rose comparably in Ep-treated and control rats during exposure to hypoxia. The data do not support the hypothesis that a rise in the plasma Ep level inhibits Ep production or secretion into the plasma.
The Pelger-Huet anomaly: a new familial association with polydactyly and trisomy 13 syndrome.
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Effect of angiotensin infusion on extrarenal erythropoietin production.
The effects of infusing subpressor doses of angiotensin II into hypoxic and anemic rats on plasma Ep levels were determined. The effect was greatest when 5 micrograms of angiotensin II per hour was infused into rats made hypoxic 18 hr after nephrectomy. Infusion of larger amounts of angiotensin II had a lesser effect on extrarenal Ep production than did infusion of 5 micrograms/hr. Infusion of angiotensin II into rats nephrectomized 1 hr prior to exposure to hypoxia affected extrarenal Ep production to a lesser degree than the infusion into rats nephrectomized 18 hr prior to hypoxia. In contrast, administration of carbon tetrachloride per os stimulated extrarenal Ep production only when nephrectomy was performed just prior to exposure to hypoxia. Administration of both CCl4 and angiotensin II to hypoxic anephric rats elevated the plasma Ep level to approximately 1.0 IRP U/ml.
The influence of age and sex on erythropoietin titers in the plasma and tissue homogenates of hypoxic rats.
Experiments were performed to determine the erythropoietin (Ep) content of homogenates of kidneys and livers of male and female rats of various ages. In all studies, homogenates were adjusted to a concentration of 4 g of tissue per 12 ml of phosphate-buffered-saline, and the stimulus to Ep production consisted of exposure to 0.42 atmosphere for 4 h. The concentration of Ep in kidneys of male rats was about three times that found in those of females and was contained predominantly in the cortical portion of the kidneys. Ep was not detectable in kidneys of rats younger than 3 weeks of age, and reached a maximum concentration after 4 weeks of age. The Ep content of the liver was barely detectable regardless of the age of the rat or its plasma Ep titer; and did not increase significantly by administering angiotensin II or CCl4 (substances which increase extrarenal Ep production).
Induction of lymphomas in mice by busulfan and chloramphenicol.
Busulfan causes long-lasting defects in the hematopoietic stem cells and in the immune system of mice. We designed studies to determine whether chloramphenicol further damaged the already defective hematopoietic stem cells of mice that were pretreated with busulfan, and we unexpectedly observed that mice given injections of the combination of busulfan and chloramphenicol developed lymphomas in relatively high incidence. The disease is invariably associated with splenomegaly and enlargement of the thymus. Leukocytosis with lymphoblasts in the peripheral blood occurred in some affected mice. The malignant cell is a lymphoblast of thymic origin. Thirteen of 37 mice which received both busulfan and chloramphenicol developed lymphomas. An additional five of the remaining 24 mice without proven lymphoma died and were not autopsied. Twelve of the 13 proven lymphomas developed within 280 days from the start of the experiment. Four of the 35 mice treated with busulfan alone developed lymphomas, and an additional five of the remaining 31 died but were not autopsied. Two of 41 mice treated with only chloramphenicol developed lymphomas. Of the mice treated with either busulfan or chloramphenicol alone that developed lymphomas, all did so more than 280 days from the start of the experiment. None of the control mice developed lymphoma. We conclude that both busulfan and chloramphenicol may induced lymphomas in mice that are not known to develop them spontaneously. The combination of both busulfan and chloramphenicol increased the frequency and accelerated the onset of the disease.
Detection of high erythropoietin titers in renal extracts of hypoxic rats.
Previous attempts to extract erythropoietin from the kidneys have not been uniformly successful and have yielded only small amounts. Attempts were therefore made to extract erythropoietin from renal extracts prepared from the supernatant obtained after homogenizing kidneys in various liquids and centrifuging at 2300 x g. Detectable amounts of erythropoietin were recovered from kidneys of nonhypoxic male but not female rats. After exposure to hypoxia, the amounts increased to levels greater than 6.0 U/kidney from males after 8 hr of hypoxia. Erythropoietin became detectable in extracts from kidneys obtained from females after 2 hr of hypoxia and rose to levels of 0.54 U/kidney after 4 hr of hypoxia. The amount of plasma trapped in the kidneys at the time of extraction was not sufficient to account for a significant amount of this erythropoietin, and erythropoietin was not detected in extracts made from livers or spleens. Severalfold larger titers of erythropoietin were detected in renal extracts, by a simple extraction procedure, than was the case in previous attempts. The reasons for this difference are discussed.
Residual marrow damage following therapy with cyclophosphamide.
Studies were performed to determine the type of residual marrow damage which occurs after injecting mice with 200 mg/kg of cyclophosphamide every 2 weeks for 5 courses. Mice treated with cyclophosphamide, and controls injected with normal saline, were studied 6 weeks after the last injection. Complete blood counts, and total nuclear cell counts from femoral marrow revealed no differences between the 2 groups. The number of CFUs in the marrow of cyclophosphamide treated mice was slightly, but significantly, lower than of controls. Cyclophosphamide treated and control mice were then exposed to 300 rad, and the rate of marrow CFUs recovery was determined. That of cyclophosphamide treated mice was significantly slower than that of controls. Stromal function of marrows from cyclophosphamide treated mice was significantly impaired. Also, however, the proliferative potential of marrow CFUs of cyclophosphamide treated mice was modestly reduced relative to that of controls. We conclude that cyclophosphamide treatment of mice results in significant residual marrow damage, due primarily to "stromal" damage, but also to decrease in the proliferative potential of CFUs.
Extraction of erythropoietin from kidneys.
Erythropoietin (Ep) in large amounts was detected in extracts of renal tissue from hypoxic rats. These extractions were performed by homogenization of kidney tissue in phosphate buffered saline, centrifugation at 3,000 g and collection of the supernate. Male kidneys contained more Ep than did females and the major portion of Ep is located in the renal cortex. Comparison of intrarenal and plasma Ep levels at various times following initiation and cessation of hypoxia appears to be a useful method for studying the kinetics of erythropoietin production and release, and also for studying feedback mechanisms that influence these functions.
Effect of short-term protein deprivation on hemopoietic functions of healthy volunteers.
To ascertain the effects of protein deprivation on hemopoietic parameters in otherwise healthy subjects, three volunteers were placed on diets containing 0.15 g protein/kg body weight for 8 days followed in 2 mo by another 8-day study period during which they ingested their usual diets containing more than 0.9 g protein/kg body weight. Complete blood counts, serum protein determinations, and tests of in vitro and in vivo leukocyte chemotaxis were performed prior to and at the conclusion of each study period. Subjects were phlebotomized of 500 ml on day 7 of each study period. Twenty-four-hour urinary erythropoietin excretion rates were assayed just prior to and again postphlebotomy. Reticulocyte counts were performed at intervals up to 1 wk postphlebotomy. Some of these determinations were replicated during a subsequent study. The hemoglobin and hematocrits decrased slightly but significantly after 8 days on low protein diets. Erythropoietin excretion rates and reticulocyte responses to phlebotomy were also less marked while subjects were on protein depleted diets. Leukocyte chemotaxis, measured both in vitro and in vivo, was also markedly reduced while subjects were on protein-depleted diets. We conclude that 8 days of moderately severe protein deprivation significantly impairs erythropoiesis and leukocyte function in otherwise healthy individuals.
Anemia in patients with chronic renal failure.
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Effect of plasma from cyclophosphamide-treated mice on CFU-S in an in vivo culture system.
Various studies suggest that humoral substances, capable of stimulating CFU-S proliferation, are released into the plasma in response to depletion of the CFU-S population by cytotoxic substances such as cyclophosphamide. To test this hypothesis, we placed 0.25 ml of a murine marrow cell suspension with an equal volume of plasma from either normal mice or from mice previously injected with 5 mg of cyclophosphamide into cellulose dialysis tubing. These tubes were then incubated in the peritoneal cavity of mice for 1-7 days. The CFU-S content of the tubes was then assayed. The CFu-S content of suspensions in normal mouse plasma declined to one-fourth of the initial value after 7 days, whereas those in plasma from mice that received cyclophosphamide 7 days previously were essentially unchanged in number. These data suggest that 7 days after injection of cyclophosphamide, the plasma contains a factor that either prevents death of CFU-S or stimulates them to proliferate. An alternative explanation is that normal plasma contains an inhibitor of CFU-S growth that is lacking in plasma of cyclophosphamide-treated mice.
Determination of plasma erythropoietin levels: an early marker of tumor activity.
A young female patient developed erythrocytosis during the third recurrence of a cerebellar hemangioblastoma. Elevated erythropoietin levels were found in the patient's plasma with normalization after resection of the tumor. High erythropoietin titers were also found in the tumor saline extract. A fourth recurrence of the tumor was heralded by a rising plasma erythropoietin level and gradual erythrocytosis despite the absence of change in the clinical picture or the brain scan. In this case, serial plasma erythropoietin determinations served as a useful early marker of the tumor activity.
Effect of carbon tetrachloride on extrarenal erythropoietin production in rats.
Liver regeneration after partial hepatectomy is associated with an increase in extrarenal Ep production; however, this surgical procedure is time-consuming and difficult to standardize. Carbon tetrachloride (CCl4) ingestion in rats is an easy and reproducible way to induce liver damage and regeneration. We therefore studied the effects of CCl4 on extrarenal Ep production in rats. Just as is the case following partial hepatectomy, extrarenal Ep production in response to hypoxia was reduced immediately after ingestion of CCl4. Thereafter it rose to supranormal levels which peaked 3 to 4 days after CCl4 ingestion (at this time Ep titers of nephrectomized, CCl4-fed rats rose to greater than 1.0 U/ml of plasma after exposure to 0.42 atmosphere for 7 hr). Extrarenal Ep production then declined, but was still supranormal 7 days after CCl4. Carbon tetrachloride did not significantly affect extrarenal Ep production in rats nephrectomized 18 hr prior to initiation of hypoxia even if they received injections of renin prior to being made hypoxic, nor did it affect Ep production in response to hypoxia in nonnephrectomized rats under the conditions used in this study.
Hematologic complications of chronic renal failure.
Uremia interferes with erythropoiesis, granulocyte, platelet, and immune functions. As a result, uremic patients are almost invariably anemic, and have a high incidence of infections and hemorrhagic complications. The anemia of renal failure, which is caused primarily by damage to the site of erythropoietin production is often complex, and complicated by hemolysis from a variety of mechanisms, iron deficiency, and so forth. Although hemodialysis ameliorates some of the hematologic complications to a variable degree, they remain a serious hinderance to the well being of this group of patients. Progress in understanding the mechanism of these problems and their therapy has been reviewed here.
Stimulation of erythropoietin secretion by single amino acids.
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Effect of protein deprivation on extrarenal erythropoietin production.
The production of erythropoietin by rats fed a protein-deficient diet is markedly decreased. This defect can be rapidly reversed by feeding protein. In the reported experiments we investigated the effect of protein deficiency on the extra-renal production of erythropoietin. The results indicate that the production of erythropoietin in nephrectomized animals is insensitive to protein depletion.