**1. Introduction**

224 Chronic Kidney Disease

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Chronic kidney disease (CKD) affects millions of people worldwide, with high incidence and prevalence and increasing costs. Anemia, a common observation in CKD, can develop in the early phases of the disease and contributes to a poor quality of life (Eknoyan *et al*., 2004).

Anemia in patients with CKD is due to many factors. Erythropoiesis and iron homeostasis are impaired as a result of a complex chain of events, including the relative deficiency of erythropoietin, chronic inflammation, blood loss, decreased iron absorption and utilization, exogenous iron and erythropoietin acquisition via biologically unregulated mechanisms (blood transfusions and medicinal erythropoietin and iron administration) (Weiss, 2009; Guidi & Santonastaso, 2010; Lankhorst & Wish, 2010).

The advent of erythropoiesis stimulating agents (ESA) and various intravenous iron preparations has resulted in a much more effective management of anemia of CKD, allowing us to maintain hemoglobin levels in certain desired ranges and to effectively treat iron deficiency. Among the emerging challenges are the risks associated with administering high ESA and iron doses, leading to elevated hemoglobin levels and iron overload (Zager *et al*., 2002).

Recombinant human erythropoietin (rHuEpo) has been available for treatment of renal disease anemia since 1989. However, rHuEpo therapy results in iron deficiency due to insufficient iron stores for the accelerated erythropoiesis. Iron deficiency is the main cause of suboptimal response to erythropoietin in dialysis patients (Cavill & Macdougall, 1993). Maintenance iron supplementation is required to successfully treat anemia; intravenous iron compounds are used to treat dialysis patients who become iron deficient.

Monitoring erythropoietin treated patients' iron status is important to detect iron deficiency and avoid the adverse effects of iron medication. The assessment of iron requirements and monitoring of therapy require accurate markers. New alternative markers for iron status that may be useful when serum ferritin and transferrin saturation are insufficient. These newer tests include reticulocyte hemoglobin content, percentage of hypochromic red cells

Assessing Iron Status in CKD Patients: New Laboratory Parameters 227

Iron stores, erythropoietic activity, hemoglobin, oxygen content, and inflammation

Essentially all circulating plasma iron normally is bound to transferrin. The liver synthesizes transferrin and secretes it into the plasma. The chelation of ferric iron serves three purposes: it renders iron soluble under physiologic conditions, it prevents iron-mediated free radical toxicity, and it facilitates transport into cells. Transferrin is the most important physiological

Although transferrin was characterized fifty years ago, its receptor eluded investigators

The molecule is a transmembrane homodimer linked by disulfide bonds. This disulfidelinked homodimer has subunits containing 760 amino acids each. Oligosaccharides account for about 5% of the 90 kDa subunit molecular mass. A broad body of literature now supports the concept that the iron-transferrin complex is internalized by receptor-mediated

Most of the body iron is associated to hemoglobin in circulating erythrocytes. Erythropoiesis is a very active process that takes place in the bone marrow and leads to the daily production of 200 billion new erythrocytes to compensate for the destruction of senescent red cells by tissue macrophages. The control of erythropoiesis depends mostly on

Macrophages play a central role in the organism as they recycle iron after phagocytosis of senescent erythrocytes. This mechanism mainly occurs in the spleen and bone marrow and

During aging, erythrocytes accumulate multiple modifications (cell shrinkage, externalization of phosphatidyl-serine, peroxydation of the membrane). The fixation and ingestion of red cells by macrophages are triggered by cellular receptor-mediated phagocytosis (through recognition of externalized phosphatidyl-serine or neoantigens of

Iron can be stored in the macrophages associated to ferritin or hemosiderin or exported to the plasma. Iron export from macrophages to transferrin is accomplished by ferroportin, the same iron-export protein as expressed in the duodenal enterocyte, and reoxydized by

Metabolically inactive iron, is stored in ferritin and hemosiderin. Normally, 95% of the stored iron in liver tissue is found in hepatocytes as ferritin. The level of serum ferritin parallels the concentration of storage iron within the body, regardless of the cell type in

The control of iron homeostasis acts at both the cellular and the systemic level and involves a complex system of different cell types, transporters, and signals. To maintain systemic iron homeostasis, communication between cells that absorb iron from the diet (duodenal enterocytes), consume iron (mainly erythroid precursors), and store iron (hepatocytes and

In the last 10 years, understanding of the regulation of iron homeostasis has changed substantially. A small peptide hormone, hepcidin, emerged as the central regulator of iron

tissue macrophages) must be tightly regulated (Swinkels *et al.*, 2006).

erythropoietin production by the kidney and on the availability of iron.

modulates the dietary iron absorption (Nemeth *et al.*, 2004).

source of iron for red cells (Ponka, 1998).

endocytosis. (Beaumont *et al.*, 2009).

senescence) (Lang *et al.*, 2005).

which it is stored.

ceruloplasmin (Knutson *et al.*, 2005).

to a lesser extent in the Küpffer cells of the liver.

until the early 1980s.

and soluble transferrin receptor, all of which have shown some promise in recent studies (Goodnough *et al*., 2010).

The percentages of hypochromic red cells (%Hypo) and reticulocyte hemoglobin content (CHr) are reported by the Siemens analyzers (Siemens Medical Solutions Diagnostics, Tarrytown NY, USA).

Two other parameters correlate to %Hypo and CHr, erythrocyte hemoglobin equivalent (RBC-He) and reticulocyte hemoglobin equivalent (Ret-He), reported by the Sysmex XE-2100 analyzer (Sysmex Corporation, Kobe, Japan); percentages of hypochromic red cells (% Hypo He) are now available on the Sysmex analyzer XE 5000 (Sysmex Corporation, Kobe, Japan.

Beckman Coulter (Beckman Coulter Inc., Miami, Fl, USA) has introduced on the LH series analysers a new parameter, low hemoglobin density (LHD%), related to the iron availability for erythropoiesis in the previous weeks; derived from mean cell hemoglobin concentration (MCHC). In this chapter the potential clinical utility of this parameter in the assessment of iron status in CKD patients is discussed.

#### **1.1 Iron homeostasis**

The normal Western diet contains 15–20 mg iron in Hem (10%) and non-Hem (ionic, 90%) forms. Only 1–2 mg of iron is absorbed and lost every day. Importantly, the total amount of iron in the body can be regulated only by absorption, whereas iron loss occurs only passively from sloughing of skin and mucosal cells as well as from blood loss. Iron absorption is balanced against iron loss so daily iron absorption may increase in response to increased iron demand (eg, growth, pregnancy or blood loss) (Conrad *et al.*, 2002; (Miret *et al.*, 2003).

Nearly all absorption of dietary iron occurs in the duodenum. Several steps are involved, including the reduction of iron to a ferrous state, apical uptake, intracellular storage or transcellular trafficking, and basolateral release. Molecular participants in each of these processes have been identified.

The non-Hem iron mainly exists in the Fe3+ state. The ferric iron is reduced to ferrous iron before it is transported across the intestinal epithelium. The reduction of iron from the ferric to the ferrous state occurs at the enterocyte brush border by means of a duodenal ferric reductase (Dcytb). Once the insoluble Fe3+ is converted to Fe2+. Ferrous iron is then transported across the apical plasma membrane of the enterocyte by divalent metal transporter 1 (DMT1) DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron, and also traffics other metal ions such as zinc, copper and cobalt by a proton-coupled mechanism (Conrad *et al.*, 2002).

Iron taken up by the enterocyte may be stored intracellularly as ferritin (and excreted in the feces when the senescent enterocyte is sloughed) or transferred across the basolateral membrane to the plasma. This iron is transferred out of the enterocyte by the basolateral transporter ferroportin; this process is facilitated by the ferroxidase activity of the ceruloplasmin homologue hephaestin (Fleming *et al.*, 2005).

There are no substantial physiologic mechanisms that regulate iron loss. Accordingly, iron homeostasis is dependent on regulatory feedback between body iron needs and intestinal iron absorption.

and soluble transferrin receptor, all of which have shown some promise in recent studies

The percentages of hypochromic red cells (%Hypo) and reticulocyte hemoglobin content (CHr) are reported by the Siemens analyzers (Siemens Medical Solutions Diagnostics,

Two other parameters correlate to %Hypo and CHr, erythrocyte hemoglobin equivalent (RBC-He) and reticulocyte hemoglobin equivalent (Ret-He), reported by the Sysmex XE-2100 analyzer (Sysmex Corporation, Kobe, Japan); percentages of hypochromic red cells (% Hypo He) are now available on the Sysmex analyzer XE 5000 (Sysmex Corporation, Kobe,

Beckman Coulter (Beckman Coulter Inc., Miami, Fl, USA) has introduced on the LH series analysers a new parameter, low hemoglobin density (LHD%), related to the iron availability for erythropoiesis in the previous weeks; derived from mean cell hemoglobin concentration (MCHC). In this chapter the potential clinical utility of this parameter in the assessment of

The normal Western diet contains 15–20 mg iron in Hem (10%) and non-Hem (ionic, 90%) forms. Only 1–2 mg of iron is absorbed and lost every day. Importantly, the total amount of iron in the body can be regulated only by absorption, whereas iron loss occurs only passively from sloughing of skin and mucosal cells as well as from blood loss. Iron absorption is balanced against iron loss so daily iron absorption may increase in response to increased iron

Nearly all absorption of dietary iron occurs in the duodenum. Several steps are involved, including the reduction of iron to a ferrous state, apical uptake, intracellular storage or transcellular trafficking, and basolateral release. Molecular participants in each of these

The non-Hem iron mainly exists in the Fe3+ state. The ferric iron is reduced to ferrous iron before it is transported across the intestinal epithelium. The reduction of iron from the ferric to the ferrous state occurs at the enterocyte brush border by means of a duodenal ferric reductase (Dcytb). Once the insoluble Fe3+ is converted to Fe2+. Ferrous iron is then transported across the apical plasma membrane of the enterocyte by divalent metal transporter 1 (DMT1) DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron, and also traffics other metal ions such as zinc, copper and cobalt by a

Iron taken up by the enterocyte may be stored intracellularly as ferritin (and excreted in the feces when the senescent enterocyte is sloughed) or transferred across the basolateral membrane to the plasma. This iron is transferred out of the enterocyte by the basolateral transporter ferroportin; this process is facilitated by the ferroxidase activity of the

There are no substantial physiologic mechanisms that regulate iron loss. Accordingly, iron homeostasis is dependent on regulatory feedback between body iron needs and intestinal

demand (eg, growth, pregnancy or blood loss) (Conrad *et al.*, 2002; (Miret *et al.*, 2003).

(Goodnough *et al*., 2010).

Tarrytown NY, USA).

**1.1 Iron homeostasis** 

processes have been identified.

iron absorption.

proton-coupled mechanism (Conrad *et al.*, 2002).

ceruloplasmin homologue hephaestin (Fleming *et al.*, 2005).

iron status in CKD patients is discussed.

Japan.

Iron stores, erythropoietic activity, hemoglobin, oxygen content, and inflammation modulates the dietary iron absorption (Nemeth *et al.*, 2004).

Essentially all circulating plasma iron normally is bound to transferrin. The liver synthesizes transferrin and secretes it into the plasma. The chelation of ferric iron serves three purposes: it renders iron soluble under physiologic conditions, it prevents iron-mediated free radical toxicity, and it facilitates transport into cells. Transferrin is the most important physiological source of iron for red cells (Ponka, 1998).

Although transferrin was characterized fifty years ago, its receptor eluded investigators until the early 1980s.

The molecule is a transmembrane homodimer linked by disulfide bonds. This disulfidelinked homodimer has subunits containing 760 amino acids each. Oligosaccharides account for about 5% of the 90 kDa subunit molecular mass. A broad body of literature now supports the concept that the iron-transferrin complex is internalized by receptor-mediated endocytosis. (Beaumont *et al.*, 2009).

Most of the body iron is associated to hemoglobin in circulating erythrocytes. Erythropoiesis is a very active process that takes place in the bone marrow and leads to the daily production of 200 billion new erythrocytes to compensate for the destruction of senescent red cells by tissue macrophages. The control of erythropoiesis depends mostly on erythropoietin production by the kidney and on the availability of iron.

Macrophages play a central role in the organism as they recycle iron after phagocytosis of senescent erythrocytes. This mechanism mainly occurs in the spleen and bone marrow and to a lesser extent in the Küpffer cells of the liver.

During aging, erythrocytes accumulate multiple modifications (cell shrinkage, externalization of phosphatidyl-serine, peroxydation of the membrane). The fixation and ingestion of red cells by macrophages are triggered by cellular receptor-mediated phagocytosis (through recognition of externalized phosphatidyl-serine or neoantigens of senescence) (Lang *et al.*, 2005).

Iron can be stored in the macrophages associated to ferritin or hemosiderin or exported to the plasma. Iron export from macrophages to transferrin is accomplished by ferroportin, the same iron-export protein as expressed in the duodenal enterocyte, and reoxydized by ceruloplasmin (Knutson *et al.*, 2005).

Metabolically inactive iron, is stored in ferritin and hemosiderin. Normally, 95% of the stored iron in liver tissue is found in hepatocytes as ferritin. The level of serum ferritin parallels the concentration of storage iron within the body, regardless of the cell type in which it is stored.

The control of iron homeostasis acts at both the cellular and the systemic level and involves a complex system of different cell types, transporters, and signals. To maintain systemic iron homeostasis, communication between cells that absorb iron from the diet (duodenal enterocytes), consume iron (mainly erythroid precursors), and store iron (hepatocytes and tissue macrophages) must be tightly regulated (Swinkels *et al.*, 2006).

In the last 10 years, understanding of the regulation of iron homeostasis has changed substantially. A small peptide hormone, hepcidin, emerged as the central regulator of iron

Assessing Iron Status in CKD Patients: New Laboratory Parameters 229

Fig. 1. Iron is absorbed from the diet by duodenal enterocytes and then bound to plasma transferrin (Tf). Fe-Tf is distributed to the bone marrow for erythropoiesis. At the end of their lifespan, senescent erythrocytes are phagocytosed by tissue macrophages and heme

Hepcidin regulates the systemic iron homeostasis; synthesized by the liver is secreted into the circulation, where it down-regulates the ferroportin-mediated release of iron from

Anemia, a common observation in CKD, can develop in the early phases of the disease is associated to poor outcomes and contributes to a reduced quality of life, with symptoms including dyspnea, headache, light-headedness, and fatigue. Anemia in patients with CKD is due to many factors. The most well-known cause is inadequate production of erythropoietin. As renal failure progresses, the contribution of erythropoietin deficiency to

Other causes which lead to impaired erythropoiesis contribute to anemia include diversion of iron traffic, diminished erythropoiesis, blunted response to erythropoietin, erythrophagocytosis, reduced proliferative activity of erythroid precursors in bone marrow, reduced survival of red cells, the decreased iron availability lead to impaired erythropoiesis

Absolute iron deficiency is defined as a decreased total iron body content. Iron deficiency anemia (IDA) occurs when iron deficiency is sufficiently severe to diminish erythropoiesis and cause the development of anemia. Functional iron deficiency describes a state where the total iron content of the body is normal or even elevated, but the iron is "locked away" and

iron is recycled back to plasma transferrin.

enterocytes, macrophages, and hepatocytes. Swinkels, D. W. et al. Clin Chem 2006;52:950-968.

anemia increases (Lankhorst & Wish, 2010).

(Weiss, 2009).

absorption, plasma iron levels, and iron distribution. Hepcidin is secreted by mainly by hepatocytes, and to a lesser extent by macrophages and adipocytes. The hormone inhibits iron flows into plasma from macrophages involved in recycling of senescent erythrocytes, duodenal enterocytes engaged in the absorption of dietary iron, and hepatocytes that store iron.( Ganz & Nemeth, 2009).

The human hepcidin gene is located on chromosome 19q13.1, encodes a precursor protein of 84 amino acids. During its export from the cytoplasm, this full-length pre-prohepcidin undergoes enzymatic cleavage, resulting in a 64 amino acids prohepcidin. Next, the 39 amino acids pro-region peptide is probably post-translationally removed, renders bioactive hepcidin-25. In human urine also are identified hepcidin-22 and hepcidin-20, which are Nterminally truncated iso-forms of hepcidin-25 (Kemna *et al.,* 2008).

Hepcidin expression is controlled by various stimuli: iron, inflammation, erythropoiesis, and hypoxia. iron and inflammation induce hepcidin production, while iron deficiency, hypoxia, and stimulation of erythropoiesis completely inhibit its production. Hepcidin is secreted into the circulation, where it down-regulates the ferroportin-mediated release of iron from enterocytes, macrophages and hepatocytes and is the key for the regulation of systemic iron homeostasis (Fleming *et al.*, 2005), reduces the quantity of circulating iron by limiting the egress of the metal from both intestinal and macrophage cells; the cellular process by which hepcidin acts, through its binding to ferroportin, thereby inducing internalization and subsequent degradation of the exporter (Bergamaschi & Villani*.*, 2009).

In the intestine, delivery of dietary iron to plasma transferrin is inhibited by increasing concentrations of hepcidin, and iron is subsequently removed from the body, through the elimination of enterocytes (desquamation process). In macrophages, degradation of ferroportin by hepcidin results in the trapping of iron inside the cells, thereby limiting the acquisition of iron by erythroid cells (Nemeth *et al.*, 2004).

Figure 1 shows and summarizes the information contained on the previous section.
