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Erythropoietin and transferrin metabolism in nephrotic syndrome
The Official Journal of the
National Kidney Foundation
AJKD
VOL 38, NO 1, JULY 2001
American Journal of Kidney Diseases
IN-DEPTH REVIEW
Erythropoietin and Transferrin Metabolism in Nephrotic Syndrome Nosratola D. Vaziri, MD ● Nephrotic syndrome is characterized by marked urinary excretion of albumin and other intermediate-size plasma proteins. This results in a profound alteration of the metabolism of many plasma proteins and protein-bound substances, as well as certain cellular and tissue proteins. This review summarizes available data on the effect of nephrotic syndrome on the metabolism and regulation of erythropoietin (EPO) and transferrin, which are essential for erythropoiesis. Studies of humans and animals have documented significant urinary losses of both EPO and transferrin in nephrotic syndrome. Urinary losses of EPO have been shown to cause EPO-deficiency anemia and prevent the normal increase in plasma EPO level in response to anemia and hypoxia in nephrotic syndrome. Similarly, transferrinuria and increased transferrin catabolism have been shown to cause hypotransferrinemia and, in some cases, iron-deficiency anemia. In addition, dissociation of iron from filtered transferrin, occasioned by a reduction in tubular fluid pH, can promote tubulointerstitial injury through the iron-catalyzed generation of oxygen free radicals. This can account in part for the role of proteinuria as a risk factor for the progression of renal disease. Subcutaneous administration of recombinant EPO has been successfully used in the management of EPOdeficiency anemia in nephrotic syndrome. Similarly, iron supplementation and nutritional support are indicated in nephrotic patients with severe transferrinuria and iron-deficiency anemia. However, correction or amelioration of the underlying proteinuria, when possible, is the ideal approach to reversal of these complications. © 2001 by the National Kidney Foundation, Inc. INDEX WORDS: Nephrotic syndrome; proteinuria; iron deficiency; anemia; erythropoietin (EPO); transferrin.
H
EAVY GLOMERULAR proteinuria is the defining feature of nephrotic syndrome. In addition to albumin, many other intermediatesize proteins are lost in the urine of humans and animals with nephrotic syndrome. Excessive urinary losses can potentially reduce plasma concentrations and produce a deficiency of the given proteins. In addition, the associated salt and water retention and volume expansion can significantly alter the volume of distribution of most proteins in nephrotic syndrome. In this regard, the volume of distribution of small- to intermediate-size proteins that have substantial extravascular distributions increases, whereas that of macromolecular species with minimal extravascular distribution can diminish or remain unchanged. Moreover, rates of biosynthesis and catabolism may increase for some proteins and decline for others. Thus, nephrotic syndrome can potentially change plasma concentrations of proteins by
causing urinary losses, as well as altering their distribution, catabolism, and biosynthesis. For example, urinary losses and depressed plasma concentrations of various hormones, hormonebinding proteins, coagulation proteins, fibrinolytic factors, immunoglobulins, enzymes, and metal-binding proteins have been shown in nephrotic humans or animals.1-14 This article reviews available data on the effects of nephrotic From the Departments of Medicine, Physiology, and Biophysics, Division of Nephrology and Hypertension, University of California, Irvine, CA. Received September 19, 2000; accepted in revised form December 22, 2000. Address reprint requests to Nostratola D. Vaziri, MD, Division of Nephrology and Hypertension, UCI Medical Center, 101 The City Dr, Orange, CA 92868. E-mail: [email protected] © 2001 by the National Kidney Foundation, Inc. 0272-6386/01/3801-0001$35.00/0 doi:10.1053/ajkd.2001.25174
American Journal of Kidney Diseases, Vol 38, No 1 (July), 2001: pp 1-8
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NOSRATOLA D. VAZIRI
syndrome on the metabolism of erythropoietin (EPO) and transferrin, which have a vital role in erythropoiesis.
EPO
Production, Regulation, and Actions of EPO EPO is a glycoprotein (molecular weight, 30.4 kd) that consists of a single strand of 165 amino acids and a large carbohydrate moiety. EPO is the product of the EPO gene, which resides on chromosome 7.15 EPO production is markedly increased by hypoxia, anemia, ascent to high altitudes, and certain hemoglobinopathies, conditions known to limit oxygen delivery to EPOproducing cells.16 Upregulation of EPO production by hypoxia recently has been shown to be mediated by a hypoxia-inducible factor (HIF-1) produced by many different cell types after exposure to hypoxia. HIF-1 serves as a general transcription factor for a number of hypoxia-induced genes, including vascular endothelial growth factor, platelet-derived growth factor, and numerous glycolytic enzymes.17 HIF-1 binds to an oxygensensitive enhancer sequence located immediately downstream from the EPO gene.18,19 Thus, HIF-1 serves as a transcription factor for EPO. EPO messenger RNA (mRNA) has been found in interstitial cells (possibly transformed fibroblasts) located near the base of proximal tubular epithelial cells in the renal cortex.20 EPO gene transcription in these cells occurs in an all-ornone manner. Consequently, the rate of EPO production at any given time depends on the number of cells expressing EPO mRNA.21 The precise manner by which hypoxia is sensed and the EPO gene is activated is not certain. However, the localization of EPO-producing cells in close vicinity to proximal tubular epithelial cells, which are highly dependent on oxygen, suggests that these cells generate the signal for transmission to the adjacent EPO-producing interstitial cells. It should be noted that small amounts of EPO are produced in nonrenal tissues, ie, hepatocytes, macrophages, and possibly erythroblasts, approximating 10% of total-body EPO production.22 Multipotential stem cells can replicate to produce either multipotential stem cells or committed unipotential progenitor cells. Accordingly,
stem cells are capable of replenishing their own pool, as well as the pools of committed unipotential progenitor cells. Replication of stem cells is primarily initiated by lineage-nonspecific growth factors, including stem-cell factor, interleukin-3, insulin growth factor, and granulocyte monocyte colony-stimulating factor. Erythroid progenitor cells appear to lose their self-renewal capacity. However, they gain EPO receptor, which promotes their replication and maturation to erythroblasts. By acquiring EPO receptor, erythroid progenitor cells lose their capacity for self-renewal, and their proliferation is directed toward a generation of readily distinguishable erythroid precursor cells. The earliest progenitor cells, ie, burst-forming erythroid cells, possess both lineage-nonspecific growth factor receptors and erythropoietin receptor. Thus, in the presence of sufficient quantities of EPO and nonspecific growth factors, they show massive proliferation, leading to the formation of many colonies of nucleated red cells.23 However, maturation of these cells to the late progenitor cells, known as colony-forming erythroid units, is coupled with a substantial loss of nonspecific growth factor receptors. Consequently, continued replication of these cells and their transformation to precursor cells primarily depends on the presence of EPO. Binding of EPO to EPO receptors (a 55-kd transmembrane protein belonging to the cytokine receptor superfamily) triggers a cascade of protein phosphorylation events that lead to the release of second messengers and cell proliferation.24 Recent in vitro studies have shown that unless EPO is present, colony-forming erythroid progenitor cells die of apoptosis. Thus, survival of these progenitor cells and their transformation to erythroid precursor cells are EPO dependent.25 After a level of maturation has been reached, the surviving colony-forming erythroid cells become activated and transform to erythroid precursor cells (erythroblasts) that are capable of synthesizing hemoglobin. In the presence of sufficient supplies of vitamin B12, folate, and iron, erythroblasts proliferate at a fixed rate and subsequently extrude their nuclei to become reticulocytes, which eventually transform to mature erythrocytes. Replication and maturation of erythroblasts appear to be independent of EPO.
ERYTHROPOIETIN AND TRANSFERRIN IN NEPHROSIS
EPO Metabolism in Nephrotic Syndrome In an earlier study, we found marked urinary excretion of EPO in a group of patients with nephrotic syndrome. Urinary losses of EPO in this population were coupled with an inappropriately low plasma EPO concentration.1 This was accompanied by a significant reduction in hematocrit in a number of patients who did not show evidence of blood loss, hemolysis, or hematopoietic factor deficiencies. We therefore concluded that the observed anemia must be caused by EPO deficiency in those instances. Thus, nephrotic syndrome was identified as a potential cause of EPO-deficiency anemia.1 In an attempt to gain further insight into the effects of nephrotic syndrome, we subsequently performed a comprehensive study of EPO metabolism, regulation, and pharmacokinetics in nephrotic and control rats.2 The study showed marked urinary EPO excretion and significant reduction of plasma EPO concentration, coupled with a mild reduction of hematocrit in nephrotic animals compared with the normal control group. Plasma EPO concentrations in the study animals were inversely related to urinary EPO excretion. Glomerular filtration rates in nephrotic animals were normal and similar to those of control rats, thus excluding renal insufficiency as a possible contributor to the associated EPO deficiency. Moreover, control animals were pair-fed with their nephrotic counterparts and maintained under identical experimental conditions to obviate possible differences in dietary intake and other conditions. Thus, the observed reduction in plasma EPO concentrations in nephrotic rats could be attributed solely to nephrotic syndrome. Results of baseline studies of nephrotic animals confirmed our earlier observations in nephrotic humans. We then compared the changes in plasma EPO concentration in response to hypobaric hypoxia and experimental anemia of different severities produced by phlebotomy and volume replacement. The experiments showed an expected robust increase in plasma EPO levels and no detectable quantities of EPO in the urine of control animals after exposure to hypobaric hypoxia. Conversely, nephrotic animals showed only a slight and insignificant increase in plasma EPO concentration, coupled with a substantial increase over baseline in urinary EPO excretion
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after a 6-hour exposure to hypobaric hypoxia. As with hypoxia, the plasma EPO response to experimentally induced anemia (hematocrits, 35%, 30%, 25%, 20%, and 15%) was negligible in nephrotic animals that instead showed progressive increases in urinary EPO excretion. Conversely, control animals showed a dramatic increase in plasma EPO concentration in response to the reduction in erythrocyte mass with no detectable urinary excretion of EPO.2 Pharmacokinetic studies performed after the intravenous injection of recombinant EPO (rEPO; 100 U/kg) showed a marked reduction in plasma EPO half-life, significant increase in rEPO clearance, and significant expansion of rEPO distribution volume in nephrotic animals compared with control animals. Moreover, urinary EPO excretion increased significantly after intravenous rEPO injection. These findings clearly showed the role of urinary excretion as a major reason for the inability of nephrotic animals to sufficiently increase plasma EPO concentration with either endogenous release or exogenous intravenous administration of EPO. We calculated the rate of endogenous EPO biosynthesis from steady-state plasma EPO level and EPO clearance data obtained from pharmacokinetic studies of animals rendered anemic by phlebotomy. Data showed a significantly lower rate of EPO biosynthesis in nephrotic animals compared with equally anemic controls. However, no significant difference was found when anemia was either absent or less severe. Thus, animal studies showed heavy urinary EPO losses, depressed plasma EPO concentrations, expanded EPO distribution (edema), increased EPO clearance, reduced plasma EPO half-life, and possibly diminished EPO biosynthesis in nephrotic syndrome.2 Treatment of EPO-Deficiency Anemia in Nephrotic Syndrome The definitive treatment of EPO-deficiency anemia in nephrotic syndrome is correction of the underlying proteinuria. This is clearly exemplified by the reported simultaneous correction of severe EPO-deficiency anemia with remission of proteinuria after a course of steroid therapy in a man with minimal change glomerulopathy.26 However, in many instances, proteinuria is unresponsive to currently available therapeutic modalities. When present, EPO-deficiency anemia
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NOSRATOLA D. VAZIRI
can be treated successfully with the regular administration of rEPO in such patients. rEPO should be administered subcutaneously to allow gradual and sustained release of the drug. Because of the short plasma half-life and rapid clearance and urinary losses of EPO, intravenous administration of rEPO is not recommended in patients with nephrotic syndrome. In an earlier report, we showed the efficacy of subcutaneous rEPO therapy in the treatment of severe disabling EPO-deficiency anemia caused by nephrotic syndrome.27 The reported efficacy of rEPO therapy in this setting was subsequently confirmed by other investigators.28 Because of the paucity of published data, established guidelines concerning rEPO therapy in patients with nephrotic syndrome have not yet been formulated. However, existing guidelines in place for rEPO therapy in patients with chronic renal failure can be used with the understanding that greater dosages may be required to cope with urinary losses of the administered rEPO. Chronic rEPO therapy can cause de novo hypertension or aggravation of preexisting hypertension29 in patients and animals with renal insufficiency. Chronic nephrotic syndrome is frequently accompanied by varying degrees of renal insufficiency, which can increase the risk for EPO-induced hypertension. Therefore, blood pressure should be monitored and hypertension should be controlled in such patients. Because nephrotic syndrome is the prototype of acquired hypercoagulable states,30 nephrotic patients should be monitored for thromboembolic complications. In this regard, rEPO therapy can increase platelet production, enhance platelet adhesion,31,32 and cause rheological modifications without significantly affecting plasma levels of coagulation factors or fibrinolytic proteins in uremic patients.32 It therefore is unlikely that EPO therapy would significantly aggravate coagulation and fibrinolytic cascade disturbances in nephrotic syndrome. Nonetheless, it is prudent to watch patients with severe nephrotic syndrome for the occurrence of thromboembolism, particularly renal vein thrombosis, regardless of the therapeutic regimen used. TRANSFERRIN
In solution, iron can serve as either an electron donor (ferrous or Fe2⫹) or electron acceptor
(ferric or Fe3⫹) element. Convenient interconvertibility of this element between the two redux states, together with its overabundance in nature, has made iron a key player in a wide array of biochemical reactions. For example, iron has an important part in numerous vital reactions, such as oxygen transport, electron transfer, DNA synthesis, and nitrogen fixation. Consequently, iron is an essential and indispensable element for life. Paradoxically, the same redox reactions responsible for the vital role of iron in various biochemical functions can be a potential source of serious cytotoxicity and tissue injury. For example, if not properly chelated, iron can serve as a powerful catalyst in single-electron redox reactions that produce cytotoxic free radicals and tissue damage. In addition, at physiological oxygen tension and pH, ferrous iron (Fe2⫹) is oxidized to ferric iron (Fe3⫹), which is readily hydrolyzed to produce insoluble products, namely, ferric hydroxide [Fe(OH)3] and oxohydroxide polymers. However, a group of highly specialized chelating molecules and sophisticated handling processes have evolved for the absorption, transport, and storage of iron to preserve solubility and prevent iron-mediated free radical generation under normal conditions. Accordingly, under normal conditions, these iron-complexing compounds virtually eliminate free iron from intracellular and extracellular compartments. Ferritin and hemosiderin (probably made of denatured ferritine) serve as the principal compounds for iron storage in various tissues. Conversely, apotransferrin serves as the primary vehicle for the transport of iron as transferrin between the sites of absorption, storage, and utilization.33 As a circulating protein, transferrin metabolism can be directly affected by proteinuria and is discussed here. Transferrin is an 80-kd monomeric glycoprotein (⬃6% carbohydrate) consisting of two homologous domains, each possessing a binding site for a ferric iron atom. Transferrin belongs to the iron-binding protein family, which includes, in addition to transferrin, the following proteins: a-lactoferrin, which is present intracellularly and secreted in milk, tears, and semen and serves as a natural bacteriostatic factor; b-ovotransferrin, present in egg white; and finally, c-melanotransferrin, a membrane-bound homologue of trans-
ERYTHROPOIETIN AND TRANSFERRIN IN NEPHROSIS
ferrin that transports iron to intracellular lowmolecular-weight ligands. Transferrin is synthesized by the liver and secreted into the circulation and extravascular space. In addition, small amounts of transferrin are produced in testicles, spleen, brain, and kidneys.34-37 Transferrin was first isolated from plasma by Schade and Caroline,38 who described it as an iron-binding component of human plasma and noted its bacteriostatic property. It has a half-life of 8 to 12 days and can bind up to two ferric iron atoms at a very high affinity (dissociation constant [Kd] ⫽ 10–23 mol/L) in a pHdependent manner.34,39-41 Binding of ferric iron to each of the two apotransferrin domains leads to a conformational change from the open to closed position. Transferrin, usually present in plasma as a mixture of diferric, monoferric, and iron-free (apoferritin) forms, is normally approximately 30% saturated with iron.33 Plasma transferrin concentrations range between 2 and 4 g/L in the healthy population. Transferrin production is upregulated by hypoxia (through HIF-1), iron deficiency, pregnancy, and estrogen and downregulated by malnutrition, inflammation, and iron overload.33 With regard to the latter, hepatic transferrin mRNA expression is inversely related to body iron stores. The primary function of transferrin is the safe transport of iron between sites of absorption, storage, and use. Accordingly, iron absorbed from the gastrointestinal tract or released from iron stores contained in macrophages is rapidly bound by transferrin in extracellular fluid. Transferrinborne iron then is delivered to the target cells for storage as ferritin or use for incorporation in heme and nonheme proteins. This phenomenon is mediated by a transferrin receptor, a 180-kd transmembrane glycoprotein homodimer consisting of two subunits linked by a disulfide bond. Transferrin receptor is expressed by all cells (except mature erythrocytes) and has the greatest affinity for diferric transferrin; it is 30-fold greater than monoferric transferrin and 500-fold greater than iron-free apotransferrin. Each transferrin receptor subunit binds one transferrin molecule on the cell surface. Subsequently, the ligandreceptor complex is internalized within the newly formed endosome, which is created from inversion of the coated pit in the plasma membrane.
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This is followed by acidification (pH ⬃5.3) of the endosomal cavity occasioned by active secretion of H⫹ by proton adenosine triphosphate pump. Endosomal pH reduction and receptor binding work in concert to facilitate the dissociation of iron from transferrin and release of free iron to the cell cytoplasm for binding by lowmolecular-weight ligands before use or storage. The iron-free transferrin-transferrin receptor complex is then returned to the cell surface and apotransferrin is released into the extracellular space.33 The amount of transferrin-borne iron (⬃3 to 4 mg) is an extremely small fraction (⬃0.1%) of total-body iron content. However, transferrin iron turnover is remarkably fast, averaging 30 mg/d or 8 to 10 times plasma iron content.33 Transferrin Metabolism in Nephrotic Syndrome Serum transferrin levels frequently are reduced in humans and animals with nephrotic syndrome.42-44 This is accompanied by and largely caused by urinary losses of this protein.12,13,45 Serum transferrin concentration is inversely related to the degree of transferrinuria in nephrotic syndrome.45 Urinary losses of transferrin can reduce serum iron concentrations and occasionally cause iron-deficiency and microcytic anemia.12,13 However, it should be noted that presumption of iron deficiency in the reported cases was based on plasma iron indices and peripheralblood investigations and not confirmed by measuring stainable iron in bone marrow or ferritin values. Because transferrin is the primary vehicle for iron delivery to erythroid cells for hemoglobin synthesis, severe hypotransferrinemia per se can cause microcytic anemia in the absence of true iron deficiency in nephrotic syndrome. In addition to adversely affecting iron metabolism, transferrinuria can potentially contribute to renal injury by promoting iron-catalyzed generation of hydroxyl radicals in renal tubules. In this regard, iron can disassociate from the filtered transferrin when luminal fluid pH decreases to approximately 6. In addition, enzymatic breakdown of the reabsorbed transferrin can liberate iron in the proximal tubular epithelial cells. The free iron released within the lumen of renal tubules or cytoplasm of tubular epithelial cells can catalyze the generation of hydroxyl radical,
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leading to tubulointerstitial injury and progressive renal disease.46,47 To my knowledge, studies correlating the degree of transferrinuria with severity of renal parenchymal injury are lacking. However, the relationship between severity of albuminuria and progression of renal disease is well established. Because urinary excretion of transferrin closely correlates with that of albumin,45 the proportional nephrotoxicity of proteinuria may be related in part to the associated transferrinuria. Specific studies are required to explore this issue. In addition to urinary losses, transferrin catabolism is significantly increased and can contribute in part to hypotransferrinemia in nephrotic syndrome.48 Although transferrin biosynthesis is frequently increased in nephrotic syndrome,48 the increase in transferrin synthesis is usually insufficient to maintain a normal plasma concentration. Increased transferrin biosynthesis in nephrotic syndrome is transcriptionally regulated and confined to the liver, with no change in transferrin expression in extrahepatic tissues.49 Malnutrition and inflammation, commonly present in nephrotic syndrome, can downregulate transferrin biosynthesis.50-52 Thus, transferrin biosynthesis may be reduced in nephrotic patients with pronounced malnutrition and/or inflammatory disorders. Treatment of Hypotransferrinemia and Iron Deficiency The definitive treatment of nephrotic syndrome–induced transferrin deficiencies is correction or amelioration of the underlying proteinuria. In addition, iron replacement and improved nutrition are required in malnourished irondepleted patients. When iron deficiency is combined with EPO deficiency, iron repletion and rEPO therapy should be instituted simultaneously. This may help reduce the theoretical risk for augmenting iron-mediated renal injury by diverting circulating transferrin toward erythroid tissue for hemoglobin synthesis as opposed to increasing the available pool of transferrin for filtration in the kidney. As noted, hypochromic microcytic anemia and reduced serum iron concentration may occur in nephrotic syndrome as a result of hypotransferrinemia as opposed to true iron deficiency. In such cases, iron supplementation may be unnecessary and ineffective.
NOSRATOLA D. VAZIRI
CONCLUSION
Nephrotic syndrome can cause massive urinary losses and altered metabolism of EPO and transferrin. This can occasionally lead to EPOand/or transferrin-deficiency anemia in nephrotic patients. Measurements of plasma EPO, transferrin, and iron concentrations, transferrin saturation, and urinary excretion of EPO and transferrin are necessary to confirm the diagnosis. In addition, patients should be evaluated for other causes of anemia, including hemolysis, blood loss, other hematopoietic factor deficiencies, and bone marrow disorders. EPO-deficiency anemia can be effectively treated with the regular subcutaneous administration of rEPO. Transferrin and iron deficiencies can be treated with iron supplementation and nutritional support. When possible, the underlying proteinuria should be controlled. REFERENCES 1. Vaziri ND, Kaupke CJ, Barton CH, Gonzales E: Plasma concentration and urinary excretion of erythropoietin in adult nephrotic syndrome. Am J Med 92:35-40, 1992 2. Zhou XJ, Vaziri ND: Erythropoietin metabolism and pharmacokinetics in experimental nephrosis. Am J Physiol Renal Physiol 263:F812-F815, 1992 3. Afrasiabi MA, Vaziri ND, Gwinup G, Mays DM, Barton CH, Ness RL, Valenta LJ: Thyroid function studies in the nephrotic syndrome. Ann Intern Med 90:335-338, 1979 4. Elias AN, Carreon G, Vaziri ND, Pandian MR, Oveisi F: The pituitary-gonadal axis in experimental nephrotic syndrome in male rats. J Lab Clin Med 120:949-954, 1992 5. Kamiseh G, Vaziri ND, Oveisi F, Ahmadnia MR, Ahmadnia L: Vitamin D absorption, plasma concentration and urinary excretion of 25-hydroxyvitamin D in nephrotic syndrome. Proc Soc Exp Biol Med 196:210-213, 1991 6. Vaziri ND, Ngo JL, Ibsen KH, Mahalwas K, Roy S, Hung EK: Deficiency and urinary losses of factor XII in adult nephrotic syndrome. Nephron 32:342-346, 1982 7. Vaziri ND, Paule P, Toohey J, Hung E, Alikhani S, Darwish R, Pahl MV: Acquired deficiency and urinary excretion of antithrombin III in nephrotic syndrome. Arch Intern Med 144:1802-1803, 1984 8. Vaziri ND, Toohey J, Paule P, Hung E, Darwish R, Barton CH, Alikhani S: Urinary excretion and deficiency of prothrombin in nephrotic syndrome. Am J Med 77:433-436, 1984 9. Vaziri ND, Gonzales EC, Shayestehfar B, Barton CH: Plasma levels and urinary excretion of fibrinolytic and protease inhibitory proteins in nephrotic syndrome. J Lab Clin Med 124:118-124, 1994 10. Vaziri ND, Gonzales E, Barton CH, Chen HT, Nguyen Q, Arquilla M: Factor XIII and its substrates, fibronectin, fibrinogen, and alpha 2-antiplasmin, in plasma and urine of patients with nephrosis. J Lab Clin Med 117:152-156, 1991 11. Falk RH, Jennette JC, Nachman PH: Nephrotic syn-
ERYTHROPOIETIN AND TRANSFERRIN IN NEPHROSIS
drome, in Brenner BM (ed): Brenner and Rector’s The Kidney. Philadelphia, PA, Saunders, 2000, pp 1266-1271 12. Rifkind D, Kravetz HM, Knight V, Schade AL: Urinary excretion of iron-binding protein in the nephrotic syndrome. N Engl J Med 256:115-118, 1961 13. Hancock DE, Onstad JW, Wolf PL: Transferrin loss into the urine with hypochromic microcytic anemia. Am J Clin Pathol 65:73-78, 1976 14. Vaziri ND, Liang K, Parks JS: Acquired lecithimcholesterol acyltransferase deficiency in nephrotic syndrome. Am J Physiol Renal Physiol 49:F823-F828, 2001 15. Egrie JC, Browne JK: The molecular biology of erythropoietin, in Erslev AJ, Adamson JW, Eschbach JW, Winearls CG (eds): Erythropoietin—Molecular, Cellular, and Clinical Biology. Baltimore, MD, Johns Hopkins University Press, 1991, pp 21-40 16. Erslev AJ, Caro J, Miller O, Silver R: Plasma erythropoietin in health and disease. Ann Clin Lab Sci 10:250-257, 1980 17. Wang GL, Semenza GL: General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci U S A 90:4304-4308, 1993 18. Beck I, Ramirez S, Weinmann R, Caro J: Enhancer element at the 3⬘ flanking region controls transcriptional response to hypoxia in the human erythropoietin gene. J Biol Chem 266:15563-15566, 1991 19. Semenza GL, Nejfelt MK, Chi SM, Antonarakis SE: Hypoxia-inducible nuclear factors bind to an enhancer element located 3⬘ to the human erythropoietin gene. Proc Natl Acad Sci U S A 88:5680-5684, 1991 20. Koury ST, Bondurant MC, Koury MJ: Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization. Blood 71:524-527, 1988 21. Koury ST, Koury MJ, Bondurant MC, Caro J, Graber SE: Quantitation of erythropoietin producing cells in kidneys of mice by in situ hybridization: Correlation with hematocrit, renal erythropoietin RNA, and serum erythropoietin concentration. Blood 74:645-651, 1989 22. Erslev AJ, Caro J, Kansu E, Silver R: Renal and extrarenal erythropoietin production in anemic rats. Br J Haematol 45:65-72, 1980 23. Stephenson JR, Axelrod AA, McLeod DL, Shreve MM: Induction of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci U S A 65:1542-1546, 1971 24. Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF: Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 80:729-738, 1995 25. Kelley LL, Koury MJ, Bondurant MC, Koury ST, Sawyer ST, Wickreng A: Survival or death of individual proerythroblast results from differing erythropoietin sensitivities. A mechanism for controlled rates of erythrocyte production. Blood 82:2340-2352, 1993 26. Yabana M, Kihara M, Toya Y, Tamura K, Matsumoto K, Takagi N, Kamijo S, Ishii M, Umemura S: Simultaneous improvement of minimal-change nephrotic syndrome and anemia with steroid therapy. Nephron 81:84-88, 1999 27. Gansevoort RT, Vaziri ND, De Jong PE: Treatment of anemia of nephrotic syndrome with recombinant erythropoietin. Am J Kidney Dis 28:274-277, 1996
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28. Ishimitsu T, Ono H, Sugiyama M, Asakawa H, Oka K, Numabe A, Abe M, Matsuoka H, Yagi S: Successful erythropoietin treatment for severe anemia in nephrotic syndrome without renal dysfunction. Nephron 74:607-610, 1996 29. Vaziri ND: Mechanism of erythropoietin-induced hypertension. Am J Kidney Dis 33:821-828, 1999 30. Vaziri ND, Barton CH: Renal vein thrombosis, in Glassock RJ (ed): Current Therapies in Nephrology and Hypertension (ed 4). St Louis, MO, Mosby, 1998, pp 269274 31. Kaupke CJ, Butler GC, Vaziri ND: Effect of recombinant human erythropoietin on platelet production in dialysis patients. J Am Soc Nephrol 3:1672-1679, 1993 32. Vaziri ND: Vascular effects of erythropoietin and anemia correction. Semin Nephrol 20:356-363, 2000 33. Ponka P, Beaumont C, Richardson DR: Function and regulation of transferrin and ferritin. Semin Hematol 35:3554, 1998 34. Schreiber G, Dryburgh H, Millership A, Matsuda Y, Inglis A, Phillips J, Edwards K, Maggs J: The synthesis and secretion of rat transferrin. J Biol Chem 254:12013-12019, 1979 35. Schaeffer E, Boisser F, Py MC, Cohen GN, Zakin MM: Cell type-specific expression of the human transferrin gene. Role of promoter, negative, and enhancer elements. J Biol Chem 264:7153-7160, 1989 36. Idzerda RL, Huebers H, Finch CA, McKnight GS: Rat transferrin gene expression: Tissue-specific regulation by iron deficiency. Proc Natl Acad Sci U S A 83:3723-3727, 1986 37. Block B, Popovici T, Chouham S, Levin MJ, Tuil D, Kahn A: Transferrin gene expression in choroid plexus of the adult rat brain. Brain Res Bull 18:573-576, 1987 38. Schade AL, Caroline L: An iron-binding component of human blood plasma. Science 104:340-343, 1946 39. Lash A, Saleem A: Iron metabolism and its regulation. A review. Ann Clin Lab Sci 25:20-30, 1995 40. Bonkovsky HL: Iron and the liver. Am J Med Sci 301:32-43, 1991 41. Andrews NC: Disorders of iron metabolism. N Engl J Med 341:1986-1995, 1999 42. Kaysen GA: Plasma composition in the nephrotic syndrome. Am J Nephrol 13:347-359, 1993 43. Warshaw BL, Check IJ, Hymes LC, DiRusso SC: Decreased serum transferrin concentration in children with the nephrotic syndrome: Effect on lymphocyte proliferation and correlation with serum immunoglobulin levels. Clin Immunol Immunopathol 33:210-219, 1984 44. Kemper MJ, Bello AB, Altrogge H, Timmermann K, Ludwig K, Muller-Wiefel DE: Iron homeostasis in relapsing steroid-sensitive nephrotic syndrome of childhood. Clin Nephrol 52:25-29, 1999 45. Howard RL, Buddington B, Alfrey AC: Urinary albumin, transferrin and iron excretion in diabetic patients. Kidney Int 40:923-926, 1991 46. Alfrey AC: Role of iron and oxygen radicals in the progression of chronic renal failure. Am J Kidney Dis 23:183-187, 1994 47. Cooper MA, Buddington B, Miller NL, Alfrey AC:
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Urinary iron speciation in nephrotic syndrome. Am J Kidney Dis 25:314-319, 1995 48. Jensen H, Bro-Jorgensen K, Jarnum S, Olesen H, Yssing M: Transferrin metabolism in the nephrotic syndrome and in protein-losing gastroenteropathy. Scand J Clin Lab Invest 21:293-304, 1968 49. Kaysen GA, Sun X, Jones H, Martin VI, Joles JA, Tsukamoto H, Couser WG, Al-Bander H: Non-iron mediated alteration in hepatic transferrin gene expression in the nephrotic rat. Kidney Int 47:1068-1077, 1995 50. Morlese JF, Forrester T, Del Rosario M, Frazer M,
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Jahoor F: Transferrin kinetics are altered in children with severe protein-energy malnutrition. J Nutr 127:1469-1474, 1997 51. Oppert M, Gleiter CH, Muller C, Reinicke A, von Ahsen N, Frei U, Eckardt KU: Kinetics and characteristics of an acute phase response following cardiac arrest. Intensive Care Med 25:1386-1394, 1999 52. Beaumier DL, Caldwell MA, Holbein BE: Inflammation triggers hypoferremia and de novo synthesis of serum transferrin and ceruloplasmin in mice. Infect Immun 46:489494, 1984
National Kidney Foundation
AJKD
VOL 38, NO 1, JULY 2001
American Journal of Kidney Diseases
IN-DEPTH REVIEW
Erythropoietin and Transferrin Metabolism in Nephrotic Syndrome Nosratola D. Vaziri, MD ● Nephrotic syndrome is characterized by marked urinary excretion of albumin and other intermediate-size plasma proteins. This results in a profound alteration of the metabolism of many plasma proteins and protein-bound substances, as well as certain cellular and tissue proteins. This review summarizes available data on the effect of nephrotic syndrome on the metabolism and regulation of erythropoietin (EPO) and transferrin, which are essential for erythropoiesis. Studies of humans and animals have documented significant urinary losses of both EPO and transferrin in nephrotic syndrome. Urinary losses of EPO have been shown to cause EPO-deficiency anemia and prevent the normal increase in plasma EPO level in response to anemia and hypoxia in nephrotic syndrome. Similarly, transferrinuria and increased transferrin catabolism have been shown to cause hypotransferrinemia and, in some cases, iron-deficiency anemia. In addition, dissociation of iron from filtered transferrin, occasioned by a reduction in tubular fluid pH, can promote tubulointerstitial injury through the iron-catalyzed generation of oxygen free radicals. This can account in part for the role of proteinuria as a risk factor for the progression of renal disease. Subcutaneous administration of recombinant EPO has been successfully used in the management of EPOdeficiency anemia in nephrotic syndrome. Similarly, iron supplementation and nutritional support are indicated in nephrotic patients with severe transferrinuria and iron-deficiency anemia. However, correction or amelioration of the underlying proteinuria, when possible, is the ideal approach to reversal of these complications. © 2001 by the National Kidney Foundation, Inc. INDEX WORDS: Nephrotic syndrome; proteinuria; iron deficiency; anemia; erythropoietin (EPO); transferrin.
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EAVY GLOMERULAR proteinuria is the defining feature of nephrotic syndrome. In addition to albumin, many other intermediatesize proteins are lost in the urine of humans and animals with nephrotic syndrome. Excessive urinary losses can potentially reduce plasma concentrations and produce a deficiency of the given proteins. In addition, the associated salt and water retention and volume expansion can significantly alter the volume of distribution of most proteins in nephrotic syndrome. In this regard, the volume of distribution of small- to intermediate-size proteins that have substantial extravascular distributions increases, whereas that of macromolecular species with minimal extravascular distribution can diminish or remain unchanged. Moreover, rates of biosynthesis and catabolism may increase for some proteins and decline for others. Thus, nephrotic syndrome can potentially change plasma concentrations of proteins by
causing urinary losses, as well as altering their distribution, catabolism, and biosynthesis. For example, urinary losses and depressed plasma concentrations of various hormones, hormonebinding proteins, coagulation proteins, fibrinolytic factors, immunoglobulins, enzymes, and metal-binding proteins have been shown in nephrotic humans or animals.1-14 This article reviews available data on the effects of nephrotic From the Departments of Medicine, Physiology, and Biophysics, Division of Nephrology and Hypertension, University of California, Irvine, CA. Received September 19, 2000; accepted in revised form December 22, 2000. Address reprint requests to Nostratola D. Vaziri, MD, Division of Nephrology and Hypertension, UCI Medical Center, 101 The City Dr, Orange, CA 92868. E-mail: [email protected] © 2001 by the National Kidney Foundation, Inc. 0272-6386/01/3801-0001$35.00/0 doi:10.1053/ajkd.2001.25174
American Journal of Kidney Diseases, Vol 38, No 1 (July), 2001: pp 1-8
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NOSRATOLA D. VAZIRI
syndrome on the metabolism of erythropoietin (EPO) and transferrin, which have a vital role in erythropoiesis.
EPO
Production, Regulation, and Actions of EPO EPO is a glycoprotein (molecular weight, 30.4 kd) that consists of a single strand of 165 amino acids and a large carbohydrate moiety. EPO is the product of the EPO gene, which resides on chromosome 7.15 EPO production is markedly increased by hypoxia, anemia, ascent to high altitudes, and certain hemoglobinopathies, conditions known to limit oxygen delivery to EPOproducing cells.16 Upregulation of EPO production by hypoxia recently has been shown to be mediated by a hypoxia-inducible factor (HIF-1) produced by many different cell types after exposure to hypoxia. HIF-1 serves as a general transcription factor for a number of hypoxia-induced genes, including vascular endothelial growth factor, platelet-derived growth factor, and numerous glycolytic enzymes.17 HIF-1 binds to an oxygensensitive enhancer sequence located immediately downstream from the EPO gene.18,19 Thus, HIF-1 serves as a transcription factor for EPO. EPO messenger RNA (mRNA) has been found in interstitial cells (possibly transformed fibroblasts) located near the base of proximal tubular epithelial cells in the renal cortex.20 EPO gene transcription in these cells occurs in an all-ornone manner. Consequently, the rate of EPO production at any given time depends on the number of cells expressing EPO mRNA.21 The precise manner by which hypoxia is sensed and the EPO gene is activated is not certain. However, the localization of EPO-producing cells in close vicinity to proximal tubular epithelial cells, which are highly dependent on oxygen, suggests that these cells generate the signal for transmission to the adjacent EPO-producing interstitial cells. It should be noted that small amounts of EPO are produced in nonrenal tissues, ie, hepatocytes, macrophages, and possibly erythroblasts, approximating 10% of total-body EPO production.22 Multipotential stem cells can replicate to produce either multipotential stem cells or committed unipotential progenitor cells. Accordingly,
stem cells are capable of replenishing their own pool, as well as the pools of committed unipotential progenitor cells. Replication of stem cells is primarily initiated by lineage-nonspecific growth factors, including stem-cell factor, interleukin-3, insulin growth factor, and granulocyte monocyte colony-stimulating factor. Erythroid progenitor cells appear to lose their self-renewal capacity. However, they gain EPO receptor, which promotes their replication and maturation to erythroblasts. By acquiring EPO receptor, erythroid progenitor cells lose their capacity for self-renewal, and their proliferation is directed toward a generation of readily distinguishable erythroid precursor cells. The earliest progenitor cells, ie, burst-forming erythroid cells, possess both lineage-nonspecific growth factor receptors and erythropoietin receptor. Thus, in the presence of sufficient quantities of EPO and nonspecific growth factors, they show massive proliferation, leading to the formation of many colonies of nucleated red cells.23 However, maturation of these cells to the late progenitor cells, known as colony-forming erythroid units, is coupled with a substantial loss of nonspecific growth factor receptors. Consequently, continued replication of these cells and their transformation to precursor cells primarily depends on the presence of EPO. Binding of EPO to EPO receptors (a 55-kd transmembrane protein belonging to the cytokine receptor superfamily) triggers a cascade of protein phosphorylation events that lead to the release of second messengers and cell proliferation.24 Recent in vitro studies have shown that unless EPO is present, colony-forming erythroid progenitor cells die of apoptosis. Thus, survival of these progenitor cells and their transformation to erythroid precursor cells are EPO dependent.25 After a level of maturation has been reached, the surviving colony-forming erythroid cells become activated and transform to erythroid precursor cells (erythroblasts) that are capable of synthesizing hemoglobin. In the presence of sufficient supplies of vitamin B12, folate, and iron, erythroblasts proliferate at a fixed rate and subsequently extrude their nuclei to become reticulocytes, which eventually transform to mature erythrocytes. Replication and maturation of erythroblasts appear to be independent of EPO.
ERYTHROPOIETIN AND TRANSFERRIN IN NEPHROSIS
EPO Metabolism in Nephrotic Syndrome In an earlier study, we found marked urinary excretion of EPO in a group of patients with nephrotic syndrome. Urinary losses of EPO in this population were coupled with an inappropriately low plasma EPO concentration.1 This was accompanied by a significant reduction in hematocrit in a number of patients who did not show evidence of blood loss, hemolysis, or hematopoietic factor deficiencies. We therefore concluded that the observed anemia must be caused by EPO deficiency in those instances. Thus, nephrotic syndrome was identified as a potential cause of EPO-deficiency anemia.1 In an attempt to gain further insight into the effects of nephrotic syndrome, we subsequently performed a comprehensive study of EPO metabolism, regulation, and pharmacokinetics in nephrotic and control rats.2 The study showed marked urinary EPO excretion and significant reduction of plasma EPO concentration, coupled with a mild reduction of hematocrit in nephrotic animals compared with the normal control group. Plasma EPO concentrations in the study animals were inversely related to urinary EPO excretion. Glomerular filtration rates in nephrotic animals were normal and similar to those of control rats, thus excluding renal insufficiency as a possible contributor to the associated EPO deficiency. Moreover, control animals were pair-fed with their nephrotic counterparts and maintained under identical experimental conditions to obviate possible differences in dietary intake and other conditions. Thus, the observed reduction in plasma EPO concentrations in nephrotic rats could be attributed solely to nephrotic syndrome. Results of baseline studies of nephrotic animals confirmed our earlier observations in nephrotic humans. We then compared the changes in plasma EPO concentration in response to hypobaric hypoxia and experimental anemia of different severities produced by phlebotomy and volume replacement. The experiments showed an expected robust increase in plasma EPO levels and no detectable quantities of EPO in the urine of control animals after exposure to hypobaric hypoxia. Conversely, nephrotic animals showed only a slight and insignificant increase in plasma EPO concentration, coupled with a substantial increase over baseline in urinary EPO excretion
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after a 6-hour exposure to hypobaric hypoxia. As with hypoxia, the plasma EPO response to experimentally induced anemia (hematocrits, 35%, 30%, 25%, 20%, and 15%) was negligible in nephrotic animals that instead showed progressive increases in urinary EPO excretion. Conversely, control animals showed a dramatic increase in plasma EPO concentration in response to the reduction in erythrocyte mass with no detectable urinary excretion of EPO.2 Pharmacokinetic studies performed after the intravenous injection of recombinant EPO (rEPO; 100 U/kg) showed a marked reduction in plasma EPO half-life, significant increase in rEPO clearance, and significant expansion of rEPO distribution volume in nephrotic animals compared with control animals. Moreover, urinary EPO excretion increased significantly after intravenous rEPO injection. These findings clearly showed the role of urinary excretion as a major reason for the inability of nephrotic animals to sufficiently increase plasma EPO concentration with either endogenous release or exogenous intravenous administration of EPO. We calculated the rate of endogenous EPO biosynthesis from steady-state plasma EPO level and EPO clearance data obtained from pharmacokinetic studies of animals rendered anemic by phlebotomy. Data showed a significantly lower rate of EPO biosynthesis in nephrotic animals compared with equally anemic controls. However, no significant difference was found when anemia was either absent or less severe. Thus, animal studies showed heavy urinary EPO losses, depressed plasma EPO concentrations, expanded EPO distribution (edema), increased EPO clearance, reduced plasma EPO half-life, and possibly diminished EPO biosynthesis in nephrotic syndrome.2 Treatment of EPO-Deficiency Anemia in Nephrotic Syndrome The definitive treatment of EPO-deficiency anemia in nephrotic syndrome is correction of the underlying proteinuria. This is clearly exemplified by the reported simultaneous correction of severe EPO-deficiency anemia with remission of proteinuria after a course of steroid therapy in a man with minimal change glomerulopathy.26 However, in many instances, proteinuria is unresponsive to currently available therapeutic modalities. When present, EPO-deficiency anemia
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NOSRATOLA D. VAZIRI
can be treated successfully with the regular administration of rEPO in such patients. rEPO should be administered subcutaneously to allow gradual and sustained release of the drug. Because of the short plasma half-life and rapid clearance and urinary losses of EPO, intravenous administration of rEPO is not recommended in patients with nephrotic syndrome. In an earlier report, we showed the efficacy of subcutaneous rEPO therapy in the treatment of severe disabling EPO-deficiency anemia caused by nephrotic syndrome.27 The reported efficacy of rEPO therapy in this setting was subsequently confirmed by other investigators.28 Because of the paucity of published data, established guidelines concerning rEPO therapy in patients with nephrotic syndrome have not yet been formulated. However, existing guidelines in place for rEPO therapy in patients with chronic renal failure can be used with the understanding that greater dosages may be required to cope with urinary losses of the administered rEPO. Chronic rEPO therapy can cause de novo hypertension or aggravation of preexisting hypertension29 in patients and animals with renal insufficiency. Chronic nephrotic syndrome is frequently accompanied by varying degrees of renal insufficiency, which can increase the risk for EPO-induced hypertension. Therefore, blood pressure should be monitored and hypertension should be controlled in such patients. Because nephrotic syndrome is the prototype of acquired hypercoagulable states,30 nephrotic patients should be monitored for thromboembolic complications. In this regard, rEPO therapy can increase platelet production, enhance platelet adhesion,31,32 and cause rheological modifications without significantly affecting plasma levels of coagulation factors or fibrinolytic proteins in uremic patients.32 It therefore is unlikely that EPO therapy would significantly aggravate coagulation and fibrinolytic cascade disturbances in nephrotic syndrome. Nonetheless, it is prudent to watch patients with severe nephrotic syndrome for the occurrence of thromboembolism, particularly renal vein thrombosis, regardless of the therapeutic regimen used. TRANSFERRIN
In solution, iron can serve as either an electron donor (ferrous or Fe2⫹) or electron acceptor
(ferric or Fe3⫹) element. Convenient interconvertibility of this element between the two redux states, together with its overabundance in nature, has made iron a key player in a wide array of biochemical reactions. For example, iron has an important part in numerous vital reactions, such as oxygen transport, electron transfer, DNA synthesis, and nitrogen fixation. Consequently, iron is an essential and indispensable element for life. Paradoxically, the same redox reactions responsible for the vital role of iron in various biochemical functions can be a potential source of serious cytotoxicity and tissue injury. For example, if not properly chelated, iron can serve as a powerful catalyst in single-electron redox reactions that produce cytotoxic free radicals and tissue damage. In addition, at physiological oxygen tension and pH, ferrous iron (Fe2⫹) is oxidized to ferric iron (Fe3⫹), which is readily hydrolyzed to produce insoluble products, namely, ferric hydroxide [Fe(OH)3] and oxohydroxide polymers. However, a group of highly specialized chelating molecules and sophisticated handling processes have evolved for the absorption, transport, and storage of iron to preserve solubility and prevent iron-mediated free radical generation under normal conditions. Accordingly, under normal conditions, these iron-complexing compounds virtually eliminate free iron from intracellular and extracellular compartments. Ferritin and hemosiderin (probably made of denatured ferritine) serve as the principal compounds for iron storage in various tissues. Conversely, apotransferrin serves as the primary vehicle for the transport of iron as transferrin between the sites of absorption, storage, and utilization.33 As a circulating protein, transferrin metabolism can be directly affected by proteinuria and is discussed here. Transferrin is an 80-kd monomeric glycoprotein (⬃6% carbohydrate) consisting of two homologous domains, each possessing a binding site for a ferric iron atom. Transferrin belongs to the iron-binding protein family, which includes, in addition to transferrin, the following proteins: a-lactoferrin, which is present intracellularly and secreted in milk, tears, and semen and serves as a natural bacteriostatic factor; b-ovotransferrin, present in egg white; and finally, c-melanotransferrin, a membrane-bound homologue of trans-
ERYTHROPOIETIN AND TRANSFERRIN IN NEPHROSIS
ferrin that transports iron to intracellular lowmolecular-weight ligands. Transferrin is synthesized by the liver and secreted into the circulation and extravascular space. In addition, small amounts of transferrin are produced in testicles, spleen, brain, and kidneys.34-37 Transferrin was first isolated from plasma by Schade and Caroline,38 who described it as an iron-binding component of human plasma and noted its bacteriostatic property. It has a half-life of 8 to 12 days and can bind up to two ferric iron atoms at a very high affinity (dissociation constant [Kd] ⫽ 10–23 mol/L) in a pHdependent manner.34,39-41 Binding of ferric iron to each of the two apotransferrin domains leads to a conformational change from the open to closed position. Transferrin, usually present in plasma as a mixture of diferric, monoferric, and iron-free (apoferritin) forms, is normally approximately 30% saturated with iron.33 Plasma transferrin concentrations range between 2 and 4 g/L in the healthy population. Transferrin production is upregulated by hypoxia (through HIF-1), iron deficiency, pregnancy, and estrogen and downregulated by malnutrition, inflammation, and iron overload.33 With regard to the latter, hepatic transferrin mRNA expression is inversely related to body iron stores. The primary function of transferrin is the safe transport of iron between sites of absorption, storage, and use. Accordingly, iron absorbed from the gastrointestinal tract or released from iron stores contained in macrophages is rapidly bound by transferrin in extracellular fluid. Transferrinborne iron then is delivered to the target cells for storage as ferritin or use for incorporation in heme and nonheme proteins. This phenomenon is mediated by a transferrin receptor, a 180-kd transmembrane glycoprotein homodimer consisting of two subunits linked by a disulfide bond. Transferrin receptor is expressed by all cells (except mature erythrocytes) and has the greatest affinity for diferric transferrin; it is 30-fold greater than monoferric transferrin and 500-fold greater than iron-free apotransferrin. Each transferrin receptor subunit binds one transferrin molecule on the cell surface. Subsequently, the ligandreceptor complex is internalized within the newly formed endosome, which is created from inversion of the coated pit in the plasma membrane.
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This is followed by acidification (pH ⬃5.3) of the endosomal cavity occasioned by active secretion of H⫹ by proton adenosine triphosphate pump. Endosomal pH reduction and receptor binding work in concert to facilitate the dissociation of iron from transferrin and release of free iron to the cell cytoplasm for binding by lowmolecular-weight ligands before use or storage. The iron-free transferrin-transferrin receptor complex is then returned to the cell surface and apotransferrin is released into the extracellular space.33 The amount of transferrin-borne iron (⬃3 to 4 mg) is an extremely small fraction (⬃0.1%) of total-body iron content. However, transferrin iron turnover is remarkably fast, averaging 30 mg/d or 8 to 10 times plasma iron content.33 Transferrin Metabolism in Nephrotic Syndrome Serum transferrin levels frequently are reduced in humans and animals with nephrotic syndrome.42-44 This is accompanied by and largely caused by urinary losses of this protein.12,13,45 Serum transferrin concentration is inversely related to the degree of transferrinuria in nephrotic syndrome.45 Urinary losses of transferrin can reduce serum iron concentrations and occasionally cause iron-deficiency and microcytic anemia.12,13 However, it should be noted that presumption of iron deficiency in the reported cases was based on plasma iron indices and peripheralblood investigations and not confirmed by measuring stainable iron in bone marrow or ferritin values. Because transferrin is the primary vehicle for iron delivery to erythroid cells for hemoglobin synthesis, severe hypotransferrinemia per se can cause microcytic anemia in the absence of true iron deficiency in nephrotic syndrome. In addition to adversely affecting iron metabolism, transferrinuria can potentially contribute to renal injury by promoting iron-catalyzed generation of hydroxyl radicals in renal tubules. In this regard, iron can disassociate from the filtered transferrin when luminal fluid pH decreases to approximately 6. In addition, enzymatic breakdown of the reabsorbed transferrin can liberate iron in the proximal tubular epithelial cells. The free iron released within the lumen of renal tubules or cytoplasm of tubular epithelial cells can catalyze the generation of hydroxyl radical,
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leading to tubulointerstitial injury and progressive renal disease.46,47 To my knowledge, studies correlating the degree of transferrinuria with severity of renal parenchymal injury are lacking. However, the relationship between severity of albuminuria and progression of renal disease is well established. Because urinary excretion of transferrin closely correlates with that of albumin,45 the proportional nephrotoxicity of proteinuria may be related in part to the associated transferrinuria. Specific studies are required to explore this issue. In addition to urinary losses, transferrin catabolism is significantly increased and can contribute in part to hypotransferrinemia in nephrotic syndrome.48 Although transferrin biosynthesis is frequently increased in nephrotic syndrome,48 the increase in transferrin synthesis is usually insufficient to maintain a normal plasma concentration. Increased transferrin biosynthesis in nephrotic syndrome is transcriptionally regulated and confined to the liver, with no change in transferrin expression in extrahepatic tissues.49 Malnutrition and inflammation, commonly present in nephrotic syndrome, can downregulate transferrin biosynthesis.50-52 Thus, transferrin biosynthesis may be reduced in nephrotic patients with pronounced malnutrition and/or inflammatory disorders. Treatment of Hypotransferrinemia and Iron Deficiency The definitive treatment of nephrotic syndrome–induced transferrin deficiencies is correction or amelioration of the underlying proteinuria. In addition, iron replacement and improved nutrition are required in malnourished irondepleted patients. When iron deficiency is combined with EPO deficiency, iron repletion and rEPO therapy should be instituted simultaneously. This may help reduce the theoretical risk for augmenting iron-mediated renal injury by diverting circulating transferrin toward erythroid tissue for hemoglobin synthesis as opposed to increasing the available pool of transferrin for filtration in the kidney. As noted, hypochromic microcytic anemia and reduced serum iron concentration may occur in nephrotic syndrome as a result of hypotransferrinemia as opposed to true iron deficiency. In such cases, iron supplementation may be unnecessary and ineffective.
NOSRATOLA D. VAZIRI
CONCLUSION
Nephrotic syndrome can cause massive urinary losses and altered metabolism of EPO and transferrin. This can occasionally lead to EPOand/or transferrin-deficiency anemia in nephrotic patients. Measurements of plasma EPO, transferrin, and iron concentrations, transferrin saturation, and urinary excretion of EPO and transferrin are necessary to confirm the diagnosis. In addition, patients should be evaluated for other causes of anemia, including hemolysis, blood loss, other hematopoietic factor deficiencies, and bone marrow disorders. EPO-deficiency anemia can be effectively treated with the regular subcutaneous administration of rEPO. Transferrin and iron deficiencies can be treated with iron supplementation and nutritional support. When possible, the underlying proteinuria should be controlled. REFERENCES 1. Vaziri ND, Kaupke CJ, Barton CH, Gonzales E: Plasma concentration and urinary excretion of erythropoietin in adult nephrotic syndrome. Am J Med 92:35-40, 1992 2. Zhou XJ, Vaziri ND: Erythropoietin metabolism and pharmacokinetics in experimental nephrosis. Am J Physiol Renal Physiol 263:F812-F815, 1992 3. Afrasiabi MA, Vaziri ND, Gwinup G, Mays DM, Barton CH, Ness RL, Valenta LJ: Thyroid function studies in the nephrotic syndrome. Ann Intern Med 90:335-338, 1979 4. Elias AN, Carreon G, Vaziri ND, Pandian MR, Oveisi F: The pituitary-gonadal axis in experimental nephrotic syndrome in male rats. J Lab Clin Med 120:949-954, 1992 5. Kamiseh G, Vaziri ND, Oveisi F, Ahmadnia MR, Ahmadnia L: Vitamin D absorption, plasma concentration and urinary excretion of 25-hydroxyvitamin D in nephrotic syndrome. Proc Soc Exp Biol Med 196:210-213, 1991 6. Vaziri ND, Ngo JL, Ibsen KH, Mahalwas K, Roy S, Hung EK: Deficiency and urinary losses of factor XII in adult nephrotic syndrome. Nephron 32:342-346, 1982 7. Vaziri ND, Paule P, Toohey J, Hung E, Alikhani S, Darwish R, Pahl MV: Acquired deficiency and urinary excretion of antithrombin III in nephrotic syndrome. Arch Intern Med 144:1802-1803, 1984 8. Vaziri ND, Toohey J, Paule P, Hung E, Darwish R, Barton CH, Alikhani S: Urinary excretion and deficiency of prothrombin in nephrotic syndrome. Am J Med 77:433-436, 1984 9. Vaziri ND, Gonzales EC, Shayestehfar B, Barton CH: Plasma levels and urinary excretion of fibrinolytic and protease inhibitory proteins in nephrotic syndrome. J Lab Clin Med 124:118-124, 1994 10. Vaziri ND, Gonzales E, Barton CH, Chen HT, Nguyen Q, Arquilla M: Factor XIII and its substrates, fibronectin, fibrinogen, and alpha 2-antiplasmin, in plasma and urine of patients with nephrosis. J Lab Clin Med 117:152-156, 1991 11. Falk RH, Jennette JC, Nachman PH: Nephrotic syn-
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drome, in Brenner BM (ed): Brenner and Rector’s The Kidney. Philadelphia, PA, Saunders, 2000, pp 1266-1271 12. Rifkind D, Kravetz HM, Knight V, Schade AL: Urinary excretion of iron-binding protein in the nephrotic syndrome. N Engl J Med 256:115-118, 1961 13. Hancock DE, Onstad JW, Wolf PL: Transferrin loss into the urine with hypochromic microcytic anemia. Am J Clin Pathol 65:73-78, 1976 14. Vaziri ND, Liang K, Parks JS: Acquired lecithimcholesterol acyltransferase deficiency in nephrotic syndrome. Am J Physiol Renal Physiol 49:F823-F828, 2001 15. Egrie JC, Browne JK: The molecular biology of erythropoietin, in Erslev AJ, Adamson JW, Eschbach JW, Winearls CG (eds): Erythropoietin—Molecular, Cellular, and Clinical Biology. Baltimore, MD, Johns Hopkins University Press, 1991, pp 21-40 16. Erslev AJ, Caro J, Miller O, Silver R: Plasma erythropoietin in health and disease. Ann Clin Lab Sci 10:250-257, 1980 17. Wang GL, Semenza GL: General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci U S A 90:4304-4308, 1993 18. Beck I, Ramirez S, Weinmann R, Caro J: Enhancer element at the 3⬘ flanking region controls transcriptional response to hypoxia in the human erythropoietin gene. J Biol Chem 266:15563-15566, 1991 19. Semenza GL, Nejfelt MK, Chi SM, Antonarakis SE: Hypoxia-inducible nuclear factors bind to an enhancer element located 3⬘ to the human erythropoietin gene. Proc Natl Acad Sci U S A 88:5680-5684, 1991 20. Koury ST, Bondurant MC, Koury MJ: Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization. Blood 71:524-527, 1988 21. Koury ST, Koury MJ, Bondurant MC, Caro J, Graber SE: Quantitation of erythropoietin producing cells in kidneys of mice by in situ hybridization: Correlation with hematocrit, renal erythropoietin RNA, and serum erythropoietin concentration. Blood 74:645-651, 1989 22. Erslev AJ, Caro J, Kansu E, Silver R: Renal and extrarenal erythropoietin production in anemic rats. Br J Haematol 45:65-72, 1980 23. Stephenson JR, Axelrod AA, McLeod DL, Shreve MM: Induction of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci U S A 65:1542-1546, 1971 24. Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF: Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 80:729-738, 1995 25. Kelley LL, Koury MJ, Bondurant MC, Koury ST, Sawyer ST, Wickreng A: Survival or death of individual proerythroblast results from differing erythropoietin sensitivities. A mechanism for controlled rates of erythrocyte production. Blood 82:2340-2352, 1993 26. Yabana M, Kihara M, Toya Y, Tamura K, Matsumoto K, Takagi N, Kamijo S, Ishii M, Umemura S: Simultaneous improvement of minimal-change nephrotic syndrome and anemia with steroid therapy. Nephron 81:84-88, 1999 27. Gansevoort RT, Vaziri ND, De Jong PE: Treatment of anemia of nephrotic syndrome with recombinant erythropoietin. Am J Kidney Dis 28:274-277, 1996
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28. Ishimitsu T, Ono H, Sugiyama M, Asakawa H, Oka K, Numabe A, Abe M, Matsuoka H, Yagi S: Successful erythropoietin treatment for severe anemia in nephrotic syndrome without renal dysfunction. Nephron 74:607-610, 1996 29. Vaziri ND: Mechanism of erythropoietin-induced hypertension. Am J Kidney Dis 33:821-828, 1999 30. Vaziri ND, Barton CH: Renal vein thrombosis, in Glassock RJ (ed): Current Therapies in Nephrology and Hypertension (ed 4). St Louis, MO, Mosby, 1998, pp 269274 31. Kaupke CJ, Butler GC, Vaziri ND: Effect of recombinant human erythropoietin on platelet production in dialysis patients. J Am Soc Nephrol 3:1672-1679, 1993 32. Vaziri ND: Vascular effects of erythropoietin and anemia correction. Semin Nephrol 20:356-363, 2000 33. Ponka P, Beaumont C, Richardson DR: Function and regulation of transferrin and ferritin. Semin Hematol 35:3554, 1998 34. Schreiber G, Dryburgh H, Millership A, Matsuda Y, Inglis A, Phillips J, Edwards K, Maggs J: The synthesis and secretion of rat transferrin. J Biol Chem 254:12013-12019, 1979 35. Schaeffer E, Boisser F, Py MC, Cohen GN, Zakin MM: Cell type-specific expression of the human transferrin gene. Role of promoter, negative, and enhancer elements. J Biol Chem 264:7153-7160, 1989 36. Idzerda RL, Huebers H, Finch CA, McKnight GS: Rat transferrin gene expression: Tissue-specific regulation by iron deficiency. Proc Natl Acad Sci U S A 83:3723-3727, 1986 37. Block B, Popovici T, Chouham S, Levin MJ, Tuil D, Kahn A: Transferrin gene expression in choroid plexus of the adult rat brain. Brain Res Bull 18:573-576, 1987 38. Schade AL, Caroline L: An iron-binding component of human blood plasma. Science 104:340-343, 1946 39. Lash A, Saleem A: Iron metabolism and its regulation. A review. Ann Clin Lab Sci 25:20-30, 1995 40. Bonkovsky HL: Iron and the liver. Am J Med Sci 301:32-43, 1991 41. Andrews NC: Disorders of iron metabolism. N Engl J Med 341:1986-1995, 1999 42. Kaysen GA: Plasma composition in the nephrotic syndrome. Am J Nephrol 13:347-359, 1993 43. Warshaw BL, Check IJ, Hymes LC, DiRusso SC: Decreased serum transferrin concentration in children with the nephrotic syndrome: Effect on lymphocyte proliferation and correlation with serum immunoglobulin levels. Clin Immunol Immunopathol 33:210-219, 1984 44. Kemper MJ, Bello AB, Altrogge H, Timmermann K, Ludwig K, Muller-Wiefel DE: Iron homeostasis in relapsing steroid-sensitive nephrotic syndrome of childhood. Clin Nephrol 52:25-29, 1999 45. Howard RL, Buddington B, Alfrey AC: Urinary albumin, transferrin and iron excretion in diabetic patients. Kidney Int 40:923-926, 1991 46. Alfrey AC: Role of iron and oxygen radicals in the progression of chronic renal failure. Am J Kidney Dis 23:183-187, 1994 47. Cooper MA, Buddington B, Miller NL, Alfrey AC:
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Urinary iron speciation in nephrotic syndrome. Am J Kidney Dis 25:314-319, 1995 48. Jensen H, Bro-Jorgensen K, Jarnum S, Olesen H, Yssing M: Transferrin metabolism in the nephrotic syndrome and in protein-losing gastroenteropathy. Scand J Clin Lab Invest 21:293-304, 1968 49. Kaysen GA, Sun X, Jones H, Martin VI, Joles JA, Tsukamoto H, Couser WG, Al-Bander H: Non-iron mediated alteration in hepatic transferrin gene expression in the nephrotic rat. Kidney Int 47:1068-1077, 1995 50. Morlese JF, Forrester T, Del Rosario M, Frazer M,
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Jahoor F: Transferrin kinetics are altered in children with severe protein-energy malnutrition. J Nutr 127:1469-1474, 1997 51. Oppert M, Gleiter CH, Muller C, Reinicke A, von Ahsen N, Frei U, Eckardt KU: Kinetics and characteristics of an acute phase response following cardiac arrest. Intensive Care Med 25:1386-1394, 1999 52. Beaumier DL, Caldwell MA, Holbein BE: Inflammation triggers hypoferremia and de novo synthesis of serum transferrin and ceruloplasmin in mice. Infect Immun 46:489494, 1984