**1.6 Leukocyte dysfunction in chronic kidney disease**

There is a complex state of leukocyte dysfunction in chronic kidney disease patients. The most important contributing factors are metabolic and functional abnormalities of leukocytes caused by the accumulation of uremic toxins that inhibit leukocyte function. In patients on dialysis, another factor influencing leukocyte function is bioincompatibility of the dialysis procedure resulting in a dysfunctional inflammatory activation (Lundberg et al. 1994; Vanholder et al. 1996; Cohen et al. 2001; Horl 2001; Cohen et al. 2003; Cheung et al. 2008).

In chronic kidney disease, there is an altered leukocyte adherence to endothelial cells, decreased activation of inflammatory cells, impaired phagocytosis and chemotaxis and an altered generation of reactive oxygen species and hydrogen peroxide (Gibbons et al. 1990; Haag-Weber & Horl 1996b; Horl 2001). Chemokine and cytokine dysregulation in chronic kidney disease gives rise to a dysfunctional activation of the immune system (Descamps-Latscha 1993; Malaponte et al. 2007; Carrero et al. 2008).

The comorbidity of the patient, such as a state of malnutrition and other chronic diseases, also plays an important role in this non-physiological inflammatory activity (Cohen et al. 1997; Stenvinkel et al. 2000; Pecoits-Filho et al. 2002). A study from our group has demonstrated that neutrophils and monocytes from patients with advanced chronic kidney disease have an impaired expression of CD11b in the interstitium compared with the corresponding cells from healthy subjects (Dadfar et al. 2004b, 2004a). The same result has been demonstrated for patients on peritoneal dialysis (Dadfar et al. 2004c).

### **1.6.1 Uremic toxins with effects on leukocytes**

There are several uremic toxins that inhibit neutrophil functions, e.g. guanidino compounds, granulocyte inhibitory protein I and II, degranulation inhibitory protein I and II (identified as angiogenin and complement factor D), κ- and λ-light chains and chemotaxis inhibitory protein (Vanholder et al. 1994b; Haag-Weber & Horl 1996a; Kaysen 2001; Horl 2002; Kaysen & Kumar 2003; Cohen & Horl 2009b, 2009a).

### **1.6.2 Patients on hemodialysis**

180 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

**Cytokines Functions References** 

Produced by macrophages and monocytes in acute and chronic inflammation. Proapoptotic. Up-regulates adhesion molecules on endothelial cells. Chemotactic factor for monocytes and primes cells for phagocytosis.

(Idriss & Naismith 2000; Janeway & Travers 2005)

(Adams & Lloyd 1997; Pupim et al. 2004; Pecoits-Filho et al. 2002; Panichi et

al. 2004)

Increases vascular permeability and vasodilatation, promotes intravascular coagulation, and causes the septic syndrome and failure of vital

important role in acute inflammation and production of acute phase proteins from

organs.

the liver.

There is a complex state of leukocyte dysfunction in chronic kidney disease patients. The most important contributing factors are metabolic and functional abnormalities of leukocytes caused by the accumulation of uremic toxins that inhibit leukocyte function. In patients on dialysis, another factor influencing leukocyte function is bioincompatibility of the dialysis procedure resulting in a dysfunctional inflammatory activation (Lundberg et al. 1994; Vanholder et al. 1996; Cohen et al. 2001; Horl 2001; Cohen et al. 2003; Cheung et al. 2008). In chronic kidney disease, there is an altered leukocyte adherence to endothelial cells, decreased activation of inflammatory cells, impaired phagocytosis and chemotaxis and an altered generation of reactive oxygen species and hydrogen peroxide (Gibbons et al. 1990; Haag-Weber & Horl 1996b; Horl 2001). Chemokine and cytokine dysregulation in chronic kidney disease gives rise to a dysfunctional activation of the immune system (Descamps-

The comorbidity of the patient, such as a state of malnutrition and other chronic diseases, also plays an important role in this non-physiological inflammatory activity (Cohen et al. 1997; Stenvinkel et al. 2000; Pecoits-Filho et al. 2002). A study from our group has demonstrated that neutrophils and monocytes from patients with advanced chronic kidney disease have an impaired expression of CD11b in the interstitium compared with the corresponding cells from healthy subjects (Dadfar et al. 2004b, 2004a). The same result has

There are several uremic toxins that inhibit neutrophil functions, e.g. guanidino compounds, granulocyte inhibitory protein I and II, degranulation inhibitory protein I and II (identified

been demonstrated for patients on peritoneal dialysis (Dadfar et al. 2004c).

**IL-6** Inflammatory marker,

**1.6 Leukocyte dysfunction in chronic kidney disease** 

Latscha 1993; Malaponte et al. 2007; Carrero et al. 2008).

**1.6.1 Uremic toxins with effects on leukocytes** 

Table 2. Cytokines analyzed in our study and their respective functions.

**TNF-α (tumor necrosis factor-α)**

> Historically, dialysis has contributed to saving many lives over the years. Without dialysis, a uremic patient unavoidably goes towards death. However, the life quality of patients on dialysis still has to be improved to develop an optimal treatment. In spite of the process in the last years to strive towards more biocompatible materials and methods, including highflux dialysis treatment, patients on hemodialysis still display a high morbidity and mortality in infections (Bloembergen & Port 1996; Powe et al. 1999; Graff et al. 2002). Neutrophil dysfunction in dialysis patients is manifested by reduced chemotaxis, adherence, respiratory burst and glucose consumption in response to an inflammatory stimulus (Vanholder et al. 1993b; Vanholder et al. 1993a).

> The dysfunctional state of inflammatory activation seen in dialysis patients could be caused by several different factors (Cheung et al. 1989; Haag-Weber et al. 1991; Descamps-Latscha 1993; Schindler et al. 2001; Carracedo et al. 2002; Horl 2002; Raj et al. 2002; Kosch et al. 2003; Koller et al. 2004). Fragments of bacterial products can be present in small amounts in the dialysate and enter the circulation by diffusion through the dialysis membrane (Horl 2002). These bacterial fragments activate proinflammatory cytokines such as IL-6, TNF-α and IL-1. There is also direct activation of complement factors and of leukocytes by contact with the dialysis membrane. Another aspect is the removal of cytokines and other inflammatory markers (lipopolysaccharide fragments, granulocyte inhibitory proteins 1 and 2, IL-1, TNFα) and complement factors (C3a, C5a) by the hemodialysis procedure as well as the adsorption of substances to the hydrophobic high-flux membrane (e.g. factor D) (Clark et al. 1999; Schindler et al. 2006). Dialysis can reduce leukocyte-endothelial interactions and impair transmigration (Thylen et al. 1997). In patients on hemodialysis with cuprophane or polysulfone membranes, a significantly higher serum level of MCP-1 is seen compared with healthy subjects both before and after the hemodialysis session, independent of the membrane used (Jacobson et al. 2000; Thylen et al. 2000).

> Biocompatibility of dialysis membranes probably plays an important role in determining leukocyte function in patients on hemodialysis (Himmelfarb et al. 1991; Himmelfarb et al. 1993; Hernandez et al. 2004; Schindler et al. 2006). High serum levels of cytokines and chemokines have been observed in patients on hemodialysis with modified cellulose membranes (Descamps-Latscha 1993; Pawlak et al. 2004; Muniz-Junqueira et al. 2005). Highflux hemodialysis causes lower levels of IL-6 and IL-1β than low-flux hemodialysis or dialysis with cuprophane membranes (Schindler et al. 2006). Our group has previously demonstrated that neutrophils and monocytes recruited to an induced interstitial inflammatory site in patients treated with low-flux bioincompatible hemodialysis have an impaired capacity of mobilizing CD11b in response to the induced inflammation, compared with the corresponding cells from healthy subjects (Thylen et al. 2000; Jacobson et al. 2002).

> Chronic kidney disease is a state that induces apoptosis, but this is normalized with continuous and high-flux hemodialysis modalities (D'Intini et al. 2004; Bordoni et al. 2006). This is in accordance with studies showing that dialysis membrane characteristics affect leukocyte cell apoptosis (Martin-Malo et al. 2000; Sela et al. 2005; Sardenberg et al. 2006). The degree of spontaneous apoptosis of leukocytes is higher when bioincompatible membranes

The MPO-study (Membrane Permeability Outcome) was a European randomized clinical trial on the effect of high-flux treatment in a large hemodialysis population. It was a prospective study which analyzed the long-term effects of membrane permeability on clinical outcomes such as mortality, morbidity, vascular access survival and nutritional status. The authors of the MPO-study did not find any significant survival benefit overall by high-flux hemodialysis versus low-flux hemodialysis. However, for some dialysis populations with low serum albumin and for patients with diabetes mellitus, a significantly lower mortality rate was observed using high-flux hemodialysis as compared with low-flux

**2. Leukocyte functional studies in patients on high-flux biocompatible** 

hemodialysis/hemodiafiltration, compared with healthy subjects (Olsson et al. 2009).

The method used was the skin chamber technique, which is well documented and has been used by a number of investigators to study transmigration and recruitment of leukocytes at the inflammatory site (Scheja & Forsgren 1985; Follin 1999; Thylen et al. 2000; Jacobson et al. 2002; Theilgaard-Monch et al. 2004; Dadfar et al. 2007; Paulsson et al. 2007). With the skin chamber technique, we measured leukocyte functions at time 0 (before the high-flux hemodialysis/hemodiafiltration session) and after 10 hours (within which time the highflux hemodialysis/hemodiafiltration treatment was performed). The terms intermediate and intense inflammation were used to designate the blister stimulated with buffer and with

Leukocytes were measured with flow cytometry or FACS (fluorescence-activated cell sorting) a method in which cells are scanned by a laser and recognized as different cell populations through their light-scattering properties. Different leukocyte populations (lymphocytes, monocytes and neutrophils) can thus be counted and expressed as a percentage of the total leukocyte population. Mean fluorescence intensity (MFI) values for the different analyses of cell functions (CD11b expression, hydrogen peroxide formation and

The CD11b expression on leukocytes, both unstimulated and after stimulation with fMLP, was studied through immunostaining. Analysis of leukocyte hydrogen peroxide formation, after stimulation with fMLP or PMA, was performed using the 2', 7'-dichlorofluorescein diacetate (DCFH-DA) method. We also stained leukocytes with Annexin V and propidium

iodide (PI) to identify cells that were in an early or late apoptotic state.

Our research group has described functions of *in vivo* extravasated monocytes and neutrophils from patients on high-flux hemodialysis/hemodiafiltration and healthy subjects (Olsson et al. 2007). The objective was to study leukocyte function and specifically, to study the up-regulation of CD11b, production of hydrogen peroxide and apoptosis of *in vivo* extravasated monocytes and neutrophils at the site of an induced interstitial inflammation in patients on high-flux hemodialysis/hemodiafiltration, compared with healthy subjects. Our group has also described the concentrations of important inflammatory mediators for neutrophils (IL-8 and MMP-9/NGAL) and monocytes (MCP-1 and MIP-1α) in the peripheral circulation and at sites of interstitial inflammation in patients on high-flux

hemodialysis (Locatelli et al. 2009).

**2.1 Methods for leukocyte functional studies** 

apoptosis) can also be measured and quantified.

autologous serum, respectively.

**hemodialysis** 

are used for hemodialysis, than when biocompatible membranes are used (Martin-Malo et al. 2000). This higher apoptotic activity in leukocytes is probably due to an antibodydependent activation of the complement system caused by the material or structure of the dialysis filters. It has been shown that heat-inactivation of complement components results in significantly lower apoptosis rates and that bioincompatible membranes cause a higher degree of apoptosis than biocompatible membranes (Koller et al. 2004).

The dialysis membrane permeability and flux are also of importance in determining the acute and chronic effects of hemodialysis on the inflammatory system. High-flux polysulfone dialysis, as opposed to low-flux polysulfone and cuprophane treatment, has been shown to improve the transmigration of circulating neutrophils (Moshfegh et al. 2002). High-flux dialysis membranes decrease the levels of the two degranulation inhibitory proteins (angiogenin and complement factor D), which could contribute to the maintained respiratory burst and phagocytic capacity seen in patients on high-flux hemodialysis (Horl 2002). There are several molecules, mainly middle-sized molecules, that are cleared to a greater extent by convective therapies, such as hemofiltration or hemodiafiltration (Clark et al. 1999). Postdilution hemofiltration was the first convective therapy used, and this method provides a high clearance of middle- and large-sized molecules but a lower clearance of small molecules. Through predilution hemofiltration, with on-line ultrafiltration, the clearance of small molecules increased substantially. In hemodiafiltration, convection is combined with diffusion, and with this mechanism the clearance of small-, middle- and large-sized molecules can be achieved to more or less the same extent (Ledebo 1998).

A number of previous studies have suggested that the type of dialysis membrane (low-flux or high-flux) is associated with differences in long-term outcome of patients undergoing hemodialysis, both in terms of morbidity and mortality (Hornberger et al. 1992; Woods & Nandakumar 2000; Cheung et al. 2003; Locatelli 2003; Chauveau et al. 2005; Canaud et al. 2006). However, the results have been conflictive regarding different outcomes.

The HEMO study, which was the first large randomized clinical trial on patient outcome depending on membrane permeability, failed to show any difference in all-cause mortality between high-flux and low-flux hemodialysis, except in some subgroups of patients (Eknoyan et al. 2002; Cheung et al. 2003; Rocco et al. 2005). Some criticism regarding the generalizability of the results from the HEMO study has been raised (Locatelli 2003). Important results from the HEMO study indicate that middle-sized molecules, e.g. parathyreoid hormone, β2-microglobulin, advanced glycosylation end products, granulocyte inhibitory proteins, advanced lipoxidation end products, advanced oxidation protein products and leptin (Horl 2002) are associated with systemic toxicity and that their accumulation predisposes dialysis patients to severe infections. An increased clearance of these molecules, e.g. β2-microglobulin, by high-flux hemodialysis is associated with a lower mortality by infectious disease (Cheung et al. 2008). An increased removal of middle-sized molecules could also have positive effects of the cardiovascular system (Vanholder et al. 2001; Vanholder et al. 2008).

In a Cochrane database review by Rabindranath et al. in 2006, the authors were unable to demonstrate a significant advantage with convective therapies over low-flux hemodialysis with regard to clinical outcomes such as mortality, dialysis-related hypotension and hospitalization (Rabindranath et al. 2006).

The DOPPS study (Dialysis Outcomes and Practice Patterns Study) revealed that patients on high-flux hemodiafiltration had a 35 % lower mortality rate than patients on low-flux hemodialysis (Canaud et al. 2006; Canaud et al. 2008).

are used for hemodialysis, than when biocompatible membranes are used (Martin-Malo et al. 2000). This higher apoptotic activity in leukocytes is probably due to an antibodydependent activation of the complement system caused by the material or structure of the dialysis filters. It has been shown that heat-inactivation of complement components results in significantly lower apoptosis rates and that bioincompatible membranes cause a higher

The dialysis membrane permeability and flux are also of importance in determining the acute and chronic effects of hemodialysis on the inflammatory system. High-flux polysulfone dialysis, as opposed to low-flux polysulfone and cuprophane treatment, has been shown to improve the transmigration of circulating neutrophils (Moshfegh et al. 2002). High-flux dialysis membranes decrease the levels of the two degranulation inhibitory proteins (angiogenin and complement factor D), which could contribute to the maintained respiratory burst and phagocytic capacity seen in patients on high-flux hemodialysis (Horl 2002). There are several molecules, mainly middle-sized molecules, that are cleared to a greater extent by convective therapies, such as hemofiltration or hemodiafiltration (Clark et al. 1999). Postdilution hemofiltration was the first convective therapy used, and this method provides a high clearance of middle- and large-sized molecules but a lower clearance of small molecules. Through predilution hemofiltration, with on-line ultrafiltration, the clearance of small molecules increased substantially. In hemodiafiltration, convection is combined with diffusion, and with this mechanism the clearance of small-, middle- and

large-sized molecules can be achieved to more or less the same extent (Ledebo 1998).

2006). However, the results have been conflictive regarding different outcomes.

2001; Vanholder et al. 2008).

hospitalization (Rabindranath et al. 2006).

hemodialysis (Canaud et al. 2006; Canaud et al. 2008).

A number of previous studies have suggested that the type of dialysis membrane (low-flux or high-flux) is associated with differences in long-term outcome of patients undergoing hemodialysis, both in terms of morbidity and mortality (Hornberger et al. 1992; Woods & Nandakumar 2000; Cheung et al. 2003; Locatelli 2003; Chauveau et al. 2005; Canaud et al.

The HEMO study, which was the first large randomized clinical trial on patient outcome depending on membrane permeability, failed to show any difference in all-cause mortality between high-flux and low-flux hemodialysis, except in some subgroups of patients (Eknoyan et al. 2002; Cheung et al. 2003; Rocco et al. 2005). Some criticism regarding the generalizability of the results from the HEMO study has been raised (Locatelli 2003). Important results from the HEMO study indicate that middle-sized molecules, e.g. parathyreoid hormone, β2-microglobulin, advanced glycosylation end products, granulocyte inhibitory proteins, advanced lipoxidation end products, advanced oxidation protein products and leptin (Horl 2002) are associated with systemic toxicity and that their accumulation predisposes dialysis patients to severe infections. An increased clearance of these molecules, e.g. β2-microglobulin, by high-flux hemodialysis is associated with a lower mortality by infectious disease (Cheung et al. 2008). An increased removal of middle-sized molecules could also have positive effects of the cardiovascular system (Vanholder et al.

In a Cochrane database review by Rabindranath et al. in 2006, the authors were unable to demonstrate a significant advantage with convective therapies over low-flux hemodialysis with regard to clinical outcomes such as mortality, dialysis-related hypotension and

The DOPPS study (Dialysis Outcomes and Practice Patterns Study) revealed that patients on high-flux hemodiafiltration had a 35 % lower mortality rate than patients on low-flux

degree of apoptosis than biocompatible membranes (Koller et al. 2004).

The MPO-study (Membrane Permeability Outcome) was a European randomized clinical trial on the effect of high-flux treatment in a large hemodialysis population. It was a prospective study which analyzed the long-term effects of membrane permeability on clinical outcomes such as mortality, morbidity, vascular access survival and nutritional status. The authors of the MPO-study did not find any significant survival benefit overall by high-flux hemodialysis versus low-flux hemodialysis. However, for some dialysis populations with low serum albumin and for patients with diabetes mellitus, a significantly lower mortality rate was observed using high-flux hemodialysis as compared with low-flux hemodialysis (Locatelli et al. 2009).
