**1.2 Anemia in CKD**

Anemia of chronic disease (ACD), the most frequent anemia among hospitalized patients, occurs in chronic inflammatory disorders, such as chronic infections, cancer and autoimmune diseases; is a hypoproliferative anemia, defined by low plasma iron concentrations in the presence of high reticuloendotelial iron stores. Cytokines are implicated in the ACD increasing iron sequestration in the reticuloendothelial system (Weiss & Goodnough, 2005), results in hyposideremia. This results in limited availability of iron for erythroid progenitor cells and iron restricted erythropoiesis.

A particular case of ACD is represented by anemia of chronic kidney disease (CKD).

CKD is becoming a major public health problem worldwide; the incidence and prevalence of this disease is increasing and the costs of treatment lead to a large burden for the health care systems, particularly in developing countries (Guidi & Santonastaso, 2010).

The severity of kidney disease is classified into five stages according to the glomerular filtration rate (GFR). It is estimated that approximately half of the patients in stage 3 CKD (GFR: 30–59 mL/min/1.73 m2) are anemic (Eknoyan *et al*., 2004).

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

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 N-

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

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

A particular case of ACD is represented by anemia of chronic kidney disease (CKD).

care systems, particularly in developing countries (Guidi & Santonastaso, 2010).

CKD is becoming a major public health problem worldwide; the incidence and prevalence of this disease is increasing and the costs of treatment lead to a large burden for the health

The severity of kidney disease is classified into five stages according to the glomerular filtration rate (GFR). It is estimated that approximately half of the patients in stage 3 CKD

Anemia of chronic disease (ACD), the most frequent anemia among hospitalized patients, occurs in chronic inflammatory disorders, such as chronic infections, cancer and autoimmune diseases; is a hypoproliferative anemia, defined by low plasma iron concentrations in the presence of high reticuloendotelial iron stores. Cytokines are implicated in the ACD increasing iron sequestration in the reticuloendothelial system (Weiss & Goodnough, 2005), results in hyposideremia. This results in limited availability of iron for

terminally truncated iso-forms of hepcidin-25 (Kemna *et al.,* 2008).

acquisition of iron by erythroid cells (Nemeth *et al.*, 2004).

erythroid progenitor cells and iron restricted erythropoiesis.

(GFR: 30–59 mL/min/1.73 m2) are anemic (Eknoyan *et al*., 2004).

iron.( Ganz & Nemeth, 2009).

**1.2 Anemia in CKD** 

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 iron is recycled back to plasma transferrin.

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 enterocytes, macrophages, and hepatocytes.

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

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 anemia increases (Lankhorst & Wish, 2010).

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 (Weiss, 2009).

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

Assessing Iron Status in CKD Patients: New Laboratory Parameters 231

< 20% and serum ferritin is < 100 ng/mL. Functional iron deficiency may be more difficult to diagnose since iron status parameters may indicate adequate iron stores. There are different criteria in defining functional iron deficiency, one of them is published by the Kidney Disease

The serum ferritin reflects storage iron, and absolute iron deficiency, according to the K/DOQI guidelines, correlates with serum ferritin <100 ng/mL. Absolute iron deficiency, the iron deficiency that is characterized by low or absent bone marrow staining for iron, is to be distinguished from functional or relative iron deficiency, which is defined as a response to intravenous iron with an increase in hemoglobin (Hb) or a decrease in erythropoiesis-

In 2004, European Best Practice Guidelines suggested an Hb target of 110 g/L (Locatelli *et al*., 2004); values of >140 g/L were considered undesirable in general, and the limit for patients with cardiovascular disease was set at 120 g/dL. Caution of not exceeding the value of Hb concentrations 120 g/L was recommended to be given also for patients with diabetes,

Assessment of anemia should include the laboratory measurement of the following

Red blood cell indices (mean cell volume MCV, mean cell hemoglobin MCH), to assess

The question regarding anemia therapy in those patients is which are the best parameters to assess the iron available for erythropoiesis. New laboratory parameters are reported by different manufacturers as potential tools for anemia and iron restricted erythropoiesis diagnosis. These tests include reticulocyte hemoglobin content, percentage of hypochromic

Serum transferrin receptor (sTfR) is a useful test for this purpose because it is not affected by inflammation so is a reliable marker of iron deficiency in mixed situations (Punnonen *et al*.,

The sTfR test is based on the fact that erythroblasts in the bone marrow will increase the presentation of membrane transferrin receptor in the setting of iron deficiency. If a patient is not receiving sufficient iron and erythropoiesis is being stimulated by an ESA, then increased transferrin receptors will become expressed on the erythroblasts, some of which come off and will be detectable in the circulation. The sTfR correlates with this membrane expression of the

red cells and soluble transferrin receptor (Wish, 2006; Goodnough *et al*., 2010).

Outcomes Quality Initiative- K/DOQI (Eknoyan *et al*. 2001).

especially if they had concurrent peripheral vascular disease.

absolute reticulocyte count , to assess erythropoietic activity

Hb concentration, to assess the degree of anemia

plasma ferritin concentration, to assess iron stores

Plasma C reactive protein, to assess inflammation

**1.2.2 New parameters for the diagnosis of anemia** 

 To assess iron available for erythropoiesis percentage of hypochromic red cells plasma transferrin Saturation reticulocyte hemoglobin content

stimulating agent requirement.

the type of anemia

1997; Beguin, 2003; Skikne, 2008).

parameters:

unavailable for the production of red blood cells. This condition is observed mainly in patients with chronic renal failure who are on hemodialysis.

Functional iron deficiency is defined as an imbalance between the iron needs for erythropoiesis and the iron supply, with the latter not maintained at sufficient rate for adequate hemoglobinization of reticulocytes and mature erythrocytes (Cavil & Macdougal, 1993).

In iron deficiency anemia (IDA) iron supply depends on the quantity of iron storage in the body, while in functional iron deficiency (iron restricted erythropoiesis) supply depends on the rate of mobilization of iron from the stores. The diagnosis of iron deficiency or functional iron deficiency is particularly challenging in patients with acute or chronic inflammatory conditions because most of the biochemical markers for iron metabolism are affected by acute phase reaction. This is the case of the anemia of chronic disease (ACD) and the anemia associated to chronic renal failure (CKD).

Recombinant human erythropoietin (rHuEpo) has been available for treatment of renal disease anemia since 1989 (Esbach *et al*., 1989). However, rHuEpo therapy results in functional 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. Maintenance iron supplementation is required to successfully treat anemia. Long term orally administered iron therapy is limited by noncompliance, gastrointestinal side effects, insufficient absorption and drug interaction; intravenous iron compounds are used to treat dialysis patients who become iron deficient (Macdougal, 1995).

Monitoring erythropoietin treated patients' iron status is important to detect iron deficiency and avoid the adverse effects of iron medication (Sunder-Plassmann & Hörl, 1997; Kletzmayr *et al*., 2002; Zager *et al*., 2002).

Biochemical indicators of iron metabolism (iron levels, transferrin, transferrin saturation, ferritin) although widely used, may be influenced by the acute phase response, which complicates clinical interpretation of the test results. Serum ferritin, an indicator of iron storage but not of iron supply, is an acute phase reactant and its levels are affected by inflammation. Because cytokines are commonly increased in CKD, serum ferritin levels might not reflect true iron stores (Mast, 2001; Coyne, 2006).

Transferrin is a negative acute phase reactant, rendering the calculation of transferrin saturation unreliable in this case. Transferrin fluctuates due to the diurnal variation of serum iron and is affected by nutritional status, leading to a lack of sensitivity and specificity in assessing iron's availability (Fishbane *et al*., 1996). For these reasons, an iron deficient erythropoietic response to rHuEpo may occur despite normal serum ferritin and transferrin values.

#### **1.2.1 Guidelines for diagnosis of anemia**

After considerable review of the literature, Kidney Disease Outcomes Quality Initiative (K/DOQI) anemia work groups in 1997, 2001, and 2006 decided that the serum ferritin and the transferrin saturation (TSAT) should be the primary tools for assessing iron management in patients with anemia and chronic kidney disease, including end- stage renal disease. For patients with chronic kidney disease, absolute iron deficiency may be diagnosed when TSAT is

unavailable for the production of red blood cells. This condition is observed mainly in

Functional iron deficiency is defined as an imbalance between the iron needs for erythropoiesis and the iron supply, with the latter not maintained at sufficient rate for adequate hemoglobinization of reticulocytes and mature erythrocytes (Cavil & Macdougal,

In iron deficiency anemia (IDA) iron supply depends on the quantity of iron storage in the body, while in functional iron deficiency (iron restricted erythropoiesis) supply depends on the rate of mobilization of iron from the stores. The diagnosis of iron deficiency or functional iron deficiency is particularly challenging in patients with acute or chronic inflammatory conditions because most of the biochemical markers for iron metabolism are affected by acute phase reaction. This is the case of the anemia of chronic disease (ACD) and the anemia

Recombinant human erythropoietin (rHuEpo) has been available for treatment of renal disease anemia since 1989 (Esbach *et al*., 1989). However, rHuEpo therapy results in functional 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. Maintenance iron supplementation is required to successfully treat anemia. Long term orally administered iron therapy is limited by noncompliance, gastrointestinal side effects, insufficient absorption and drug interaction; intravenous iron compounds are used

Monitoring erythropoietin treated patients' iron status is important to detect iron deficiency and avoid the adverse effects of iron medication (Sunder-Plassmann & Hörl, 1997;

Biochemical indicators of iron metabolism (iron levels, transferrin, transferrin saturation, ferritin) although widely used, may be influenced by the acute phase response, which complicates clinical interpretation of the test results. Serum ferritin, an indicator of iron storage but not of iron supply, is an acute phase reactant and its levels are affected by inflammation. Because cytokines are commonly increased in CKD, serum ferritin levels

Transferrin is a negative acute phase reactant, rendering the calculation of transferrin saturation unreliable in this case. Transferrin fluctuates due to the diurnal variation of serum iron and is affected by nutritional status, leading to a lack of sensitivity and specificity in assessing iron's availability (Fishbane *et al*., 1996). For these reasons, an iron deficient erythropoietic response to rHuEpo may occur despite normal serum ferritin and

After considerable review of the literature, Kidney Disease Outcomes Quality Initiative (K/DOQI) anemia work groups in 1997, 2001, and 2006 decided that the serum ferritin and the transferrin saturation (TSAT) should be the primary tools for assessing iron management in patients with anemia and chronic kidney disease, including end- stage renal disease. For patients with chronic kidney disease, absolute iron deficiency may be diagnosed when TSAT is

to treat dialysis patients who become iron deficient (Macdougal, 1995).

might not reflect true iron stores (Mast, 2001; Coyne, 2006).

patients with chronic renal failure who are on hemodialysis.

associated to chronic renal failure (CKD).

Kletzmayr *et al*., 2002; Zager *et al*., 2002).

**1.2.1 Guidelines for diagnosis of anemia**

transferrin values.

1993).

< 20% and serum ferritin is < 100 ng/mL. Functional iron deficiency may be more difficult to diagnose since iron status parameters may indicate adequate iron stores. There are different criteria in defining functional iron deficiency, one of them is published by the Kidney Disease Outcomes Quality Initiative- K/DOQI (Eknoyan *et al*. 2001).

The serum ferritin reflects storage iron, and absolute iron deficiency, according to the K/DOQI guidelines, correlates with serum ferritin <100 ng/mL. Absolute iron deficiency, the iron deficiency that is characterized by low or absent bone marrow staining for iron, is to be distinguished from functional or relative iron deficiency, which is defined as a response to intravenous iron with an increase in hemoglobin (Hb) or a decrease in erythropoiesisstimulating agent requirement.

In 2004, European Best Practice Guidelines suggested an Hb target of 110 g/L (Locatelli *et al*., 2004); values of >140 g/L were considered undesirable in general, and the limit for patients with cardiovascular disease was set at 120 g/dL. Caution of not exceeding the value of Hb concentrations 120 g/L was recommended to be given also for patients with diabetes, especially if they had concurrent peripheral vascular disease.

Assessment of anemia should include the laboratory measurement of the following parameters:

	- percentage of hypochromic red cells
		- plasma transferrin Saturation
		- reticulocyte hemoglobin content
