**4. Erythrocyte senescence and/or damage**

In stage 5 CKD patients, the erythrocytes are metabolically stressed by the unfavourable plasmatic environment, due to metabolite accumulation; by the high rate of haemoglobin autoxidation, due to the increase in haemoglobin turnover, a physiologic compensation mechanism triggered to compensate anaemia (Lucchi, 2000; Stoya, 2002). The erythrocytes will be further stressed during the haemodialysis procedure. Therefore, the erythrocytes are continuously challenged to sustain haemoglobin in its reduced functional form and to maintain the integrity and deformability of the membrane.

When haemoglobin is denatured, it links to the cytoplasmic pole of band 3, triggering its aggregation and leading to the formation of strictly lipidic portions of the membrane, poorly linked to the cytoskeleton. These cells are, probably, more prone to undergo vesiculation (loss of poorly linked membrane portions) whenever they have to circulate through the haemodialysis membranes or the microvasculature. Vesiculation may, therefore, lead to modifications in the erythrocyte membrane of stage 5 CKD patients (Reliene, 2002; Rocha, 2005).

Erythrocytes that develop intracellular defects earlier during their life span are removed prematurely from circulation (Santos-Silva, 1998; Rocha-Pereira, 2004). The removal of senescent or damaged erythrocytes seems to involve the development of a senescent neoantigen on the membrane surface, marking the cell for death. This neoantigen is immunologically related to band 3 (Kay, 1994). The deterioration of the erythrocyte metabolism and/or of its antioxidant defences may lead to the development of oxidative stress within the cell, allowing oxidation and linkage of denatured haemoglobin to the cytoplasmatic domain of band 3, promoting its aggregation, the binding of natural anti-band 3 autoantibodies and complement activation, marking the erythrocyte for death. The band 3 profile [high molecular weight aggregates (HMWAg), band 3 monomer and proteolytic fragments (Pfrag)] is used in order to differentiate younger, damaged and/or senescent erythrocytes. Older and damaged erythrocytes present with higher HMWAg and lower Pfrag. Younger erythrocytes show reduced HMWAg and higher Pfrag (Santos-Silva, 1998). Several diseases, known as inflammatory conditions, present an abnormal band 3 profile, suggestive of oxidative stress development (Santos-Silva, 1998; Belo, 2002; Rocha-Pereira, 2004).

translocation of erythrocytes from the splanchnic circulation (and possibly from the splenic circulation) in order to compensate the hypovolemic stress during dialysis ultrafiltration. We also found, after haemodialysis, an increase in mean cell hemoglobin concentration and a decrease in mean cell volume that could be related to erythrocyte membrane protein loss during the hemodialysis procedure (Costa, 2008b). Markers of neutrophil activation were also found to be increased after haemodialysis. In fact, a decrease in CXCR1 neutrophil expression was observed after the haemodialysis procedure [before haemodialysis: 252.25 ± 45.14 MFI (mean fluorescence intensity) vs after haemodialysis: 239.71 ± 47.62 MFI; *p*=0.04], as well as an increase in elastase and lactoferrin plasma levels (Table 2). The enhanced neutrophil activation state after haemodialysis could result from different mechanisms; namely, complement activation, direct interaction with haemodialysis membrane, and from the passage into the blood of bacterial fragments, such as LPS, from contaminated dialysate

In stage 5 CKD patients, the erythrocytes are metabolically stressed by the unfavourable plasmatic environment, due to metabolite accumulation; by the high rate of haemoglobin autoxidation, due to the increase in haemoglobin turnover, a physiologic compensation mechanism triggered to compensate anaemia (Lucchi, 2000; Stoya, 2002). The erythrocytes will be further stressed during the haemodialysis procedure. Therefore, the erythrocytes are continuously challenged to sustain haemoglobin in its reduced functional form and to

When haemoglobin is denatured, it links to the cytoplasmic pole of band 3, triggering its aggregation and leading to the formation of strictly lipidic portions of the membrane, poorly linked to the cytoskeleton. These cells are, probably, more prone to undergo vesiculation (loss of poorly linked membrane portions) whenever they have to circulate through the haemodialysis membranes or the microvasculature. Vesiculation may, therefore, lead to modifications in the erythrocyte membrane of stage 5 CKD patients (Reliene, 2002; Rocha,

Erythrocytes that develop intracellular defects earlier during their life span are removed prematurely from circulation (Santos-Silva, 1998; Rocha-Pereira, 2004). The removal of senescent or damaged erythrocytes seems to involve the development of a senescent neoantigen on the membrane surface, marking the cell for death. This neoantigen is immunologically related to band 3 (Kay, 1994). The deterioration of the erythrocyte metabolism and/or of its antioxidant defences may lead to the development of oxidative stress within the cell, allowing oxidation and linkage of denatured haemoglobin to the cytoplasmatic domain of band 3, promoting its aggregation, the binding of natural anti-band 3 autoantibodies and complement activation, marking the erythrocyte for death. The band 3 profile [high molecular weight aggregates (HMWAg), band 3 monomer and proteolytic fragments (Pfrag)] is used in order to differentiate younger, damaged and/or senescent erythrocytes. Older and damaged erythrocytes present with higher HMWAg and lower Pfrag. Younger erythrocytes show reduced HMWAg and higher Pfrag (Santos-Silva, 1998). Several diseases, known as inflammatory conditions, present an abnormal band 3 profile, suggestive of oxidative stress development (Santos-Silva, 1998; Belo, 2002; Rocha-Pereira,

through the dialyzer membrane.

2005).

2004).

**4. Erythrocyte senescence and/or damage**

maintain the integrity and deformability of the membrane.

Leukocyte activation is part of an inflammatory response, and is an important source of ROS and proteases, both of which may impose oxidative and proteolytic damages to erythrocyte and plasma constituents. Actually, oxidative stress has been reported to occur in stage 5 CKD patients and has been proposed as a significant factor in haemodialysis-related shortened erythrocyte survival.

In literature, there are few reports about the effect of CKD and haemodialysis procedure in erythrocyte membrane protein composition (Matos, 1997; Wu, 1998; Ibrahim, 2002). Studies performed in erythrocytes from stage 5 CKD patients, using cuprophane and polyacrylonitrile dialysis membranes, showed some changes in the membrane proteins, namely, a reduction in spectrin and band 3, and an isolated reduction in band 3, respectively (Sevilhano, 1990; Delmas-Beauvieux, 1995). Wu et al (Wu, 1998) and Ibrahim et al (2002) showed that stage 5 CKD patients presented a median osmotic fragility higher than the controls, and, after the haemodialysis procedure, that osmotic fragility decreased.

Recently, we reported for the first time, changes in the erythrocyte membrane band 3 profile in stage 5 CKD patients. These patients presented a decrease in HMWAg and in HMWAg/band 3 monomer ratio (Fig. 3 and table 3). These changes seem to reflect a younger erythrocyte population; however, CKD presented also a decrease in Pfrag and in Pfrag/band 3 monomer ratio, both suggesting a rise in erythrocyte damage. Thus, it seems that the band 3 profile observed in CKD patients is associated both to an increase in younger erythrocytes and to an increase in damaged erythrocytes (Costa, 2008c). This study also showed that the haemodialysis procedure *per se* does not lead to an increase in the studied markers of erythrocyte damage. Actually, no differences were found after haemodialysis, in band 3 profile.


\* p<0.05 *v*s controls. HMWAg; high molecular weight aggregates; Pfrag: proteolytic fragments. Results are presented as mean ± standard deviation or as median (interquartile ranges).

Table 3. Band 3 profile for controls and stage 5 CKD patients.

Some changes in erythrocyte membrane protein composition of stage 5 CKD patients using high-flux polysulfone FX-class dialysers of Fresenius, were also observed (Costa, 2008b; Costa, 2008d). A decrease in spectrin was the most significant change (table 4). This reduction in spectrin may account for a poor linkage of the cytoskeleton to the membrane, favoring membrane vesiculation, and, probably, a reduction in the erythrocyte lifespan of

Neutrophil Activation and Erythrocyte Membrane

reflecting a vertical membrane protein disturbance.

Adapted from Costa, 2008b.

immediately after haemodialysis procedure.

Protein Composition in Stage 5 Chronic Kidney Disease Patients 217

these patients (Reliene, 2002). Significant increases in protein bands 6 and 7 were also observed, which may further reflect an altered membrane protein interaction and destabilization of membrane structure. This membrane destabilization was further strengthened by the significant changes observed for spectrin/band 3 ratio (Costa, 2008b; Costa, 2008d). These membrane protein changes may be due to a higher erythrocyte metabolic stress and/or to changes resulting from the haemodialysis procedure *per se*.

Studying the effect of the haemodialysis procedure on erythrocyte membrane protein composition in stage 5 CKD patients, by evaluating membrane protein composition before and immediately after haemodialysis procedure (table 5), some trends towards the control profile were observed for some of the membrane proteins – band 3, band 6 and band 7; spectrin showed an even lower value after haemodialysis, and ankyrin, protein 4.1, protein 4.2 and band 5 also presented a trend to decrease. Comparing the ratios before and after haemodialysis, only the ratio spectrin/band 3 showed a statistically significant value,

Spectrin (%) 25.58 (24.10-27.07) 24.47 (22.31-26.95)\* Ankyrin (%) 6.39 1.55 6.23 1.28 Band 3 (%) 38.10 3.78 41.13 2.44\* Protein 4.1 (%) 6.48 1.60 6.39 1.69 Protein 4.2 (%) 4.34 0.99 4.84 1.04 Band 5 (%) 6.56 0.91 6.71 0.59 Band 6 (%) 6.46 0.87 6.17 1.15 Band 7 (%) 2.09 0.43 2.37 0.34 Protein 4.1/Spectrin 0.243 0.070 0.251 0.081 Protein 4.1/Band 3 0.170 (0.138-0.206) 0.163 (0.121-0.202) Protein 4.2/Band 3 0.114 (0.101-0.133) 0.118 (0.101-0.147) Spectrin/Band 3 0.685 (0.626-0.796) 0.647 (0.566-0.689)\* Ankyrin/Band 3 0.171 0.049 0.152 0.330 Spectrin/Ankirin 4.48 1.361 4.45 1.49

\* *p*<0.05, *vs* before haemodialysis. Results are presented as mean ± standard deviation or as median (interquartile ranges). HMWAg; high molecular weight aggregates; Pfrag: proteolytic fragments.

Haemodialysis procedure seems to have an important role in the changes observed for erythrocyte membrane protein composition; however, their exact origin(s) are not yet fully understood. An hypothesis is that the increased plasma levels of elastase found in stage 5

Table 5. Erythrocyte membrane protein profile for stage 5 CKD patients, before and

**Stage 5 CKD patients (n=20) Before haemodialysis After haemodialysis**

Fig. 3. A- Illustration of two band 3 profiles, one presented by a control (C), and the other presented by a stage 5 CKD patient (P). B- Examples of densitometer tracing of immunoblots for band 3 profile, C- Control; P – stage 5 CKD patient. HMWAg; high molecular weight aggregates; Pfrag: proteolytic fragments.


Table 4. Erythrocyte membrane protein profile for controls and stage 5 CKD patients.\* *p*<0.05, *vs* controls. Results are presented as mean ± standard deviation or as median (interquartile ranges). HMWAg; high molecular weight aggregates; Pfrag: proteolytic fragments. Adapted from Costa, 2008d.

Fig. 3. A- Illustration of two band 3 profiles, one presented by a control (C), and the other

**Controls (n=26)**

Spectrin (%) 27.63 (26.41-28.79) 24.27 (19.39-26.13)\* Ankyrin (%) 6.971.62 6.53 1.90 Band 3 (%) 38.57 3.99 39.294.03 Protein 4.1 (%) 7.561.45 7.24 1.49 Protein 4.2 (%) 5.510.72 5.44 1.44 Band 5 (%) 6.820.86 6.87 1.03 Band 6 (%) 5.191.04 6.981.37\* Band 7 (%) 2.200.65 3.32 1.24\* Protein 4.1/Spectrin 0.276 ± 0.624 0.330 ± 0.120a) Protein 4.1/Band 3 0.192 (0.154–0.227) 0.183 (0.155-0.208) Protein 4.2/Band 3 0.149 (0.125-0.162) 0.138 (0.110-0.163) Spectrin/Band 3 0.707 (0.649-0.822) 0.569 (0.512 -0.686)\* Ankyrin/Band 3 0.185 ± 0.585 0.169 ± 0.057 Spectrin/Ankirin 4.18 ± 1.07 3.77 ± 1.84 Table 4. Erythrocyte membrane protein profile for controls and stage 5 CKD patients.\* *p*<0.05, *vs* controls. Results are presented as mean ± standard deviation or as median (interquartile ranges). HMWAg; high molecular weight aggregates; Pfrag: proteolytic

**CKD stage 5 Patients (n=63)**

presented by a stage 5 CKD patient (P). B- Examples of densitometer tracing of immunoblots for band 3 profile, C- Control; P – stage 5 CKD patient. HMWAg; high

molecular weight aggregates; Pfrag: proteolytic fragments.

fragments. Adapted from Costa, 2008d.

these patients (Reliene, 2002). Significant increases in protein bands 6 and 7 were also observed, which may further reflect an altered membrane protein interaction and destabilization of membrane structure. This membrane destabilization was further strengthened by the significant changes observed for spectrin/band 3 ratio (Costa, 2008b; Costa, 2008d). These membrane protein changes may be due to a higher erythrocyte metabolic stress and/or to changes resulting from the haemodialysis procedure *per se*.

Studying the effect of the haemodialysis procedure on erythrocyte membrane protein composition in stage 5 CKD patients, by evaluating membrane protein composition before and immediately after haemodialysis procedure (table 5), some trends towards the control profile were observed for some of the membrane proteins – band 3, band 6 and band 7; spectrin showed an even lower value after haemodialysis, and ankyrin, protein 4.1, protein 4.2 and band 5 also presented a trend to decrease. Comparing the ratios before and after haemodialysis, only the ratio spectrin/band 3 showed a statistically significant value, reflecting a vertical membrane protein disturbance.


\* *p*<0.05, *vs* before haemodialysis. Results are presented as mean ± standard deviation or as median (interquartile ranges). HMWAg; high molecular weight aggregates; Pfrag: proteolytic fragments. Adapted from Costa, 2008b.

Table 5. Erythrocyte membrane protein profile for stage 5 CKD patients, before and immediately after haemodialysis procedure.

Haemodialysis procedure seems to have an important role in the changes observed for erythrocyte membrane protein composition; however, their exact origin(s) are not yet fully understood. An hypothesis is that the increased plasma levels of elastase found in stage 5

Neutrophil Activation and Erythrocyte Membrane

changes.

**damage** 

Gallagher, 2008).

Protein Composition in Stage 5 Chronic Kidney Disease Patients 219

trends towards a decrease in spectrin [25.6 (25.1-26.9%) *vs* 24.7 (24.4-25.6%), *p*=0.073) and an increase in band 3 [36.6 (34.8-37.6%) *vs* 39.1 (36.9-39.4%), *p*=0.077), as compared with erythrocytes incubated without elastase. Similar changes were found for the erythrocytes incubated with 0.1 μg/mL of elastase. In non-responders stage 5 CKD patients, the erythrocytes incubated with 0.1 and 0.5 μg/mL of elastase, showed a significant decrease in spectrin [25.5 (24.9-25.9%) and 25.3 (24.8-26.2%), respectively *vs* 26.4 (26.0-27.3%), *p*=0.011 for

These findings suggest that the erythrocytes from stage 5 CKD patients, before the haemodialysis procedure, are more susceptible to the proteolytic action of elastase upon the membrane. Considering that after the haemodialysis procedure the composition of the erythrocyte membrane from stage 5 CKD patients did not change, it seems that the more susceptible erythrocytes were removed during the haemodialysis procedure. Moreover, the release of neutrophil activation products, such as elastase, during haemodialysis may contribute to the removal of the more damaged cells, by enhancing membrane protein

**5. Other pathologies associated with neutrophil activation and erythrocyte** 

Several physiological (physical exercise, pregnancy) and pathological (hereditary spherocytosis, cardiovascular disease, preeclampsia, psoriasis) conditions presenting with neutrophilic leukocytosis have been associated to an altered erythrocyte membrane protein composition and to other changes reflecting erythrocyte damage. Moreover, they have been associated to increased neutrophil activation products, suggesting that leukocyte activation

In Hereditary Spherocytosis (HS), mutations in genes encoding for some membrane proteins - band 3, spectrin, protein band 4.2 and ankyrin - may result in their partial or inaccurate assembly to the membrane. Deficiencies in one or more of those proteins cause a decrease in membrane stability that, in turn, leads to loss of membrane surface area through membrane vesiculation. By losing membrane vesicles, the cell will become spherocytic and the membrane more rigid, triggering the sequestration of cell in the spleen and, therefore, the reduction of the erythrocyte lifespan and the development of anemia (Mohandas &

Two distinct pathways lead to the reduction in membrane surface area: i) deficiencies in spectrin, ankyrin, or protein 4.2 reduce the density of the membrane cytoskeleton, causing a weaker linkage to the lipid bilayer, favoring the loss of membrane vesicles containing lipids and band 3; ii) deficiency in band 3 favors the development of band 3 deficient areas in the membrane, with loss of the lipid-stabilizing effect of band 3, and therefore, the release of band 3-free microvesicles, from the membrane (Iolascon, 2003; Perrotta, 2008). In a recent work by our group (Rocha, 2010), studying 160 HS patients, the analysis of erythrocyte membrane protein profile showed that 109 patients presented a primary deficiency in band 3, 35 patients a primary ankyrin deficiency, 14 patients an isolated deficiency in spectrin and 2 patients an isolated deficiency in protein 4.2. Furthermore, severe HS patients presented with higher neutrophil count and higher levels of TNF-α, IFN-, elastase, lactoferrin and ferritin. Our data show HS as a disease linked to enhanced erythropoiesis that is disturbed in the more severe forms, to which inflammation, at least in part, seems to contribute.

both], as compared to erythrocytes incubated without elastase (Fig. 4).

may trigger injuries in the neighboring erythrocytes.

CKD patients could induce changes in erythrocyte membrane proteins, leading to a decrease in erythrocyte lifespan, and, consequently, to an increase in the degree of the anaemia. This hypothesis was tested (Pereira, 2011), by performing some *in vitro* assays using erythrocytes from 18 stage 5 CKD patients (10 responders and 8 non-responders to recombinant human erythropoietin therapy) and from 8 healthy controls; erythrocyte suspensions in phosphate buffered saline, pH 7.4, were incubated at 37º C, under gentle rotation, in the presence of 0.03, 0.1 and 0.5 μg/mL of neutrophil elastase. These assays used erythrocytes collected before and immediately after the haemodialysis procedure.

Fig. 4. Changes in ankyrin (A), spectrin (B) and band 3 (C) observed for erythrocytes from responder stage 5 CKD patients before haemodialysis, when incubated without and with elastase; changes in spectrin presented by erythrocytes from non-responder stage 5 CKD patients before haemodialysis, when incubated without and with elastase (D). Adapted from Pereira, 2011.

No significant differences were found between the protein composition of the erythrocyte membranes from healthy controls and from stage 5 CKD patients, when their erythrocytes, collected after the haemodialysis procedure, were incubated without and with different elastase concentrations. However, the erythrocytes from stage 5 CKD patients, collected before the haemodialysis procedure, showed some susceptibility to elastase; the erythrocytes from responders stage 5 CKD patients, incubated with 0.5 μg/mL of elastase showed a significant decrease in ankyrin [7.0 (6.5-7.5%) *vs* 6.0 (5.9-6.5%), *p*=0.024], and

CKD patients could induce changes in erythrocyte membrane proteins, leading to a decrease in erythrocyte lifespan, and, consequently, to an increase in the degree of the anaemia. This hypothesis was tested (Pereira, 2011), by performing some *in vitro* assays using erythrocytes from 18 stage 5 CKD patients (10 responders and 8 non-responders to recombinant human erythropoietin therapy) and from 8 healthy controls; erythrocyte suspensions in phosphate buffered saline, pH 7.4, were incubated at 37º C, under gentle rotation, in the presence of 0.03, 0.1 and 0.5 μg/mL of neutrophil elastase. These assays used erythrocytes collected

Fig. 4. Changes in ankyrin (A), spectrin (B) and band 3 (C) observed for erythrocytes from responder stage 5 CKD patients before haemodialysis, when incubated without and with elastase; changes in spectrin presented by erythrocytes from non-responder stage 5 CKD patients before haemodialysis, when incubated without and with elastase (D). Adapted

No significant differences were found between the protein composition of the erythrocyte membranes from healthy controls and from stage 5 CKD patients, when their erythrocytes, collected after the haemodialysis procedure, were incubated without and with different elastase concentrations. However, the erythrocytes from stage 5 CKD patients, collected before the haemodialysis procedure, showed some susceptibility to elastase; the erythrocytes from responders stage 5 CKD patients, incubated with 0.5 μg/mL of elastase showed a significant decrease in ankyrin [7.0 (6.5-7.5%) *vs* 6.0 (5.9-6.5%), *p*=0.024], and

from Pereira, 2011.

before and immediately after the haemodialysis procedure.

trends towards a decrease in spectrin [25.6 (25.1-26.9%) *vs* 24.7 (24.4-25.6%), *p*=0.073) and an increase in band 3 [36.6 (34.8-37.6%) *vs* 39.1 (36.9-39.4%), *p*=0.077), as compared with erythrocytes incubated without elastase. Similar changes were found for the erythrocytes incubated with 0.1 μg/mL of elastase. In non-responders stage 5 CKD patients, the erythrocytes incubated with 0.1 and 0.5 μg/mL of elastase, showed a significant decrease in spectrin [25.5 (24.9-25.9%) and 25.3 (24.8-26.2%), respectively *vs* 26.4 (26.0-27.3%), *p*=0.011 for both], as compared to erythrocytes incubated without elastase (Fig. 4).

These findings suggest that the erythrocytes from stage 5 CKD patients, before the haemodialysis procedure, are more susceptible to the proteolytic action of elastase upon the membrane. Considering that after the haemodialysis procedure the composition of the erythrocyte membrane from stage 5 CKD patients did not change, it seems that the more susceptible erythrocytes were removed during the haemodialysis procedure. Moreover, the release of neutrophil activation products, such as elastase, during haemodialysis may contribute to the removal of the more damaged cells, by enhancing membrane protein changes.
