**4. Cell-free nucleic acids as biomarkers in dialytic process**

During hemodialysis, the interaction of dialyzer membrane with patient´s blood leads to the activation of alternate pathway of complement. The biologically active complement component C5A is generated and activates the aggregation of neutrophils and their adherence to the endothelial surfaces. Neutrophils harvested during hemodialysis exhibit altered oxidative response, chemotaxis, aggregation, and adherence (Lewis & Van Epps,

Cell-Free Nucleic Acids as Biomarkers of Biocompatibility in Dialytic Process 207

The authors calculated also very carefully the half-live of this fraction of cfDNA and its rate of clearance. Their study was limited by selection of patients with postdialytic concentrations of cfDNA high inough for performance of experiments. The half-life of cfDNA in blood of hemodialyzed patients immediately after hemodialysis was determined as 4 minutes in this study. The rate of cfDNA clearance from circulation calculated by authors is equivalent to the DNA content of approximately 15 g of a solid tissue such as liver or of the leucocyte content of

Although the nucleosomes were found in the blood of patients after hemodialysis in similar amount as in the patients with lupus erythematosus, the hemodialysis itself does not induce any autoimmune response. According to their results, Rumore et al. (1992) hypothesized that in patients with lupus erythematosus an impairment of mechanism of nucleosome

Atamaniuk et al. (2006) investigated the levels of cfDNA in hemodialyzed patients together with markers of early and late apoptosis in leucocytes. For detection of early and late apoptosis flow cytometric measurements of annexin V expression in combination with 7 amino-actinomycin D (7AAD) were used. Annnexin V has high affinity for phosphatidylserine which is translocated during early apoptosis from the inner to the outer surface of the plasma membrane. The nuclear 7AAD staining is possible only in the late apoptosis or in dead cells because the plasma membrane is desintegrated in such stages and allows the throughput of the stain into the nucleus. Employing this methodology, the leucocyte can be classified as normal (annexin V- and 7AAD-), early apoptotic (annexin V+ and 7AAD -), and apoptotic (annexinV + and 7AAD+). Ten patients hemodialyzed on synthetic polymer membranes [Fresenius Polysulfone Capillary dialyzers (F6) low-flux] and 30 healthy subjects were examined in the study. Blood samples from hemodialyzed patients were obtained before hemodialysis (HD), after 20 min of HD, at the end of HD. The concentrations of cfDNA in plasma samples were measured using Vistra Green and human placental DNA calibrators. The emitted fluorescent signals were measured in the

In HD patients the cfDNA levels before hemodialysis were slightly higher than in controls. The significant increase was detected after 20 min of HD. The highest values of cfDNA concentrations were determined at the end of HD. The cfDNA samples isolated from plasma of patients were subjected to agarose gel electrophoresis. In pre-HD samples and in samples

The fraction of leucocytes in early apoptosis was significantly higher in predialytic interval in patients than in controls. The number of apoptotic leucocytes increased during HD. The authors conclude that the apoptosis induced by the contact of leucocytes with dialysis membrane may be the main source of elevated concentrations of cfDNA during HD sessions. Different types of dialysis membrane were examined in the study by García-Moreira et al. (2006) performed with regard to their influence on the cfDNA levels in plasma. In this study, concentrations of cfDNA were measured first time using real-time quantitative PCR. The sequence of β*-globin* gene was amplified to quantify the total amount of cfDNA in plasma samples. To establish the range of normal values, the samples from 100 healthy

Thirty patients on regular HD were studied in 52 sessions. Chronic renal failure in patients was caused by diabetic nephropathy (7 patients), glomerulosclerosis (6 patients), nephroangioesclerosis (6 patients), chronic pyelonephritis (3 patients), polycystic kidney

taken after 20 min of HD the weaker ladders than in samples after HD were observed.

600 ml of whole blood at WBC of 5000/mm3 per day (Rumore et al., 1992).

clearance contribute to the development of autoimmune disease.

LightCycler at 530 nm and reported in picograms per microliters.

voluntary blood donors were examined.

1987). Neutrophils have short half-live - under normal conditions they spend approximately 12h in the circulation and then migrate into normal tissues or are attracted by chemotactic stimuli to inflamed tissues. Once in tissues, they do not return back to circulation but undergo apoptosis and are cleared by phagocytosis (Savill 1997).

Direct contact of cell with membranes in dialyzer can also induce apoptosis (Carracedo et al., 1995). Interaction of cell-surface protein of monocytes with dialysis membrane can stimulate mononuclear cells to interact with neutrophils and induce their apoptosis (Nahar et al., 2001). Interaction between dialysis membrane and blood leads not only to complement activation but also to cytokine synthesis and release. Certain cytokines, such as tumor necrosis factor (TNF) and interleukin (IL)-1β 1, component C5a and bacterial lipopolysacchride (LPS) can increase neutrophil survival (Colotta et al., 1992; Lee et al., 1993).

As some interleukins are generated during hemodialysis and activation of monocytes occurs it seems that hemodialysis effects are comparable to the pathophysiological mechanisms of inflammation and hypersensitivity response (García-Moreira et al., 2006).

The survival of leucocytes during hemodialysis is influenced by numerous factors therefore an analyte which would mirror the complex character of biological events during hemodialysis with regard to the impact of the procedure on immune system of each individual patient may be very helpful. The study of cell-free nucleic acids seems to be a promising approach. In the next sections, the first attempts in this newly arising field will be presented.

#### **4.1 Cell-free nucleic acids in hemodialyzed patients**

The first study focused on the examination of cfDNA was carried out in 1977 by means of semiquantitative counter-immunoelectrophoresis (Steinman & Ackad, 1977). It is till today the only one study examining the appearance of cfDNA in vitro experiment using circulation of fresh blood through dialysis coil. The authors conclude that the passage of blood through the coil itself can account, at least in part, for circulating DNA and speculate about the leucocytes as the main source of this DNA.

Fournie et al. (1989) used the detection method based on incorporation of radiolabeled nucleotide in nick translation reaction and confirmed the results obtained by Steinman & Ackad (1977). In the study by Fournie and coauthors, 45 patients during 99 sessions of hemodialysis or hemofiltration were followed. Independently on the method of treatment, during the first 3 h of the session the elevations of plasma DNA were observed. The authors concluded that the dialytic procedure induces the death of leucocytes on the artificial surfaces and the release of their DNA into blood thus is responsible for elevated values of extracellular DNA. In this study plasma DNA levels were increased in interdialytic interval in 41 out of 99 samples, among samples with elevated cfDNA levels were 18 out of 24 samples collected from hepatitis B infected person.

The study performed by Rumore et al. (1992) used the same cfDNA detection method as Steinman & Ackad (1977) and exploited the possibilities of the visualization of radiolabeled DNA on electrophoretic gels to demonstrate that in the blood of hemodialyzed patients the electrophoretic patterns known from patients with lupus erythematosus (SLE) are clearly recognizable. This study documented also by immunoprecipitation that at least the large fraction of cfDNA in the blood of hemodialyzed patients circulates in the form of nucleosomes like in the patients with lupus erythematosus. The autoradiograms provided the picture of DNA samples fragmented in the typical manner with predominance of bands corresponding to DNA of 150-200, 400 and in some cases 600 bp.

1987). Neutrophils have short half-live - under normal conditions they spend approximately 12h in the circulation and then migrate into normal tissues or are attracted by chemotactic stimuli to inflamed tissues. Once in tissues, they do not return back to circulation but

Direct contact of cell with membranes in dialyzer can also induce apoptosis (Carracedo et al., 1995). Interaction of cell-surface protein of monocytes with dialysis membrane can stimulate mononuclear cells to interact with neutrophils and induce their apoptosis (Nahar et al., 2001). Interaction between dialysis membrane and blood leads not only to complement activation but also to cytokine synthesis and release. Certain cytokines, such as tumor necrosis factor (TNF) and interleukin (IL)-1β 1, component C5a and bacterial lipopolysacchride (LPS) can increase

As some interleukins are generated during hemodialysis and activation of monocytes occurs it seems that hemodialysis effects are comparable to the pathophysiological mechanisms of

The survival of leucocytes during hemodialysis is influenced by numerous factors therefore an analyte which would mirror the complex character of biological events during hemodialysis with regard to the impact of the procedure on immune system of each individual patient may be very helpful. The study of cell-free nucleic acids seems to be a promising approach. In the next sections, the first attempts in this newly arising field

The first study focused on the examination of cfDNA was carried out in 1977 by means of semiquantitative counter-immunoelectrophoresis (Steinman & Ackad, 1977). It is till today the only one study examining the appearance of cfDNA in vitro experiment using circulation of fresh blood through dialysis coil. The authors conclude that the passage of blood through the coil itself can account, at least in part, for circulating DNA and speculate

Fournie et al. (1989) used the detection method based on incorporation of radiolabeled nucleotide in nick translation reaction and confirmed the results obtained by Steinman & Ackad (1977). In the study by Fournie and coauthors, 45 patients during 99 sessions of hemodialysis or hemofiltration were followed. Independently on the method of treatment, during the first 3 h of the session the elevations of plasma DNA were observed. The authors concluded that the dialytic procedure induces the death of leucocytes on the artificial surfaces and the release of their DNA into blood thus is responsible for elevated values of extracellular DNA. In this study plasma DNA levels were increased in interdialytic interval in 41 out of 99 samples, among samples with elevated cfDNA levels were 18 out of 24

The study performed by Rumore et al. (1992) used the same cfDNA detection method as Steinman & Ackad (1977) and exploited the possibilities of the visualization of radiolabeled DNA on electrophoretic gels to demonstrate that in the blood of hemodialyzed patients the electrophoretic patterns known from patients with lupus erythematosus (SLE) are clearly recognizable. This study documented also by immunoprecipitation that at least the large fraction of cfDNA in the blood of hemodialyzed patients circulates in the form of nucleosomes like in the patients with lupus erythematosus. The autoradiograms provided the picture of DNA samples fragmented in the typical manner with predominance of bands

undergo apoptosis and are cleared by phagocytosis (Savill 1997).

inflammation and hypersensitivity response (García-Moreira et al., 2006).

neutrophil survival (Colotta et al., 1992; Lee et al., 1993).

**4.1 Cell-free nucleic acids in hemodialyzed patients** 

about the leucocytes as the main source of this DNA.

samples collected from hepatitis B infected person.

corresponding to DNA of 150-200, 400 and in some cases 600 bp.

will be presented.

The authors calculated also very carefully the half-live of this fraction of cfDNA and its rate of clearance. Their study was limited by selection of patients with postdialytic concentrations of cfDNA high inough for performance of experiments. The half-life of cfDNA in blood of hemodialyzed patients immediately after hemodialysis was determined as 4 minutes in this study. The rate of cfDNA clearance from circulation calculated by authors is equivalent to the DNA content of approximately 15 g of a solid tissue such as liver or of the leucocyte content of 600 ml of whole blood at WBC of 5000/mm3 per day (Rumore et al., 1992).

Although the nucleosomes were found in the blood of patients after hemodialysis in similar amount as in the patients with lupus erythematosus, the hemodialysis itself does not induce any autoimmune response. According to their results, Rumore et al. (1992) hypothesized that in patients with lupus erythematosus an impairment of mechanism of nucleosome clearance contribute to the development of autoimmune disease.

Atamaniuk et al. (2006) investigated the levels of cfDNA in hemodialyzed patients together with markers of early and late apoptosis in leucocytes. For detection of early and late apoptosis flow cytometric measurements of annexin V expression in combination with 7 amino-actinomycin D (7AAD) were used. Annnexin V has high affinity for phosphatidylserine which is translocated during early apoptosis from the inner to the outer surface of the plasma membrane. The nuclear 7AAD staining is possible only in the late apoptosis or in dead cells because the plasma membrane is desintegrated in such stages and allows the throughput of the stain into the nucleus. Employing this methodology, the leucocyte can be classified as normal (annexin V- and 7AAD-), early apoptotic (annexin V+ and 7AAD -), and apoptotic (annexinV + and 7AAD+). Ten patients hemodialyzed on synthetic polymer membranes [Fresenius Polysulfone Capillary dialyzers (F6) low-flux] and 30 healthy subjects were examined in the study. Blood samples from hemodialyzed patients were obtained before hemodialysis (HD), after 20 min of HD, at the end of HD. The concentrations of cfDNA in plasma samples were measured using Vistra Green and human placental DNA calibrators. The emitted fluorescent signals were measured in the LightCycler at 530 nm and reported in picograms per microliters.

In HD patients the cfDNA levels before hemodialysis were slightly higher than in controls. The significant increase was detected after 20 min of HD. The highest values of cfDNA concentrations were determined at the end of HD. The cfDNA samples isolated from plasma of patients were subjected to agarose gel electrophoresis. In pre-HD samples and in samples taken after 20 min of HD the weaker ladders than in samples after HD were observed.

The fraction of leucocytes in early apoptosis was significantly higher in predialytic interval in patients than in controls. The number of apoptotic leucocytes increased during HD. The authors conclude that the apoptosis induced by the contact of leucocytes with dialysis membrane may be the main source of elevated concentrations of cfDNA during HD sessions.

Different types of dialysis membrane were examined in the study by García-Moreira et al. (2006) performed with regard to their influence on the cfDNA levels in plasma. In this study, concentrations of cfDNA were measured first time using real-time quantitative PCR. The sequence of β*-globin* gene was amplified to quantify the total amount of cfDNA in plasma samples. To establish the range of normal values, the samples from 100 healthy voluntary blood donors were examined.

Thirty patients on regular HD were studied in 52 sessions. Chronic renal failure in patients was caused by diabetic nephropathy (7 patients), glomerulosclerosis (6 patients), nephroangioesclerosis (6 patients), chronic pyelonephritis (3 patients), polycystic kidney

Cell-Free Nucleic Acids as Biomarkers of Biocompatibility in Dialytic Process 209

The study of cell-free nucleic acids in connection with hemodialysis promises new clinically important findings. Application of new methods – mainly the technology of Next Generation Sequencing (NGS) in combination with bionformatics – will bring huge amount of qualitative and quantitative data and allow the study of tiny fractions of specific sequences on the total circulating nucleic acids background. Recently, no studies focused on analysis of microRNA and mRNA in the plasma of hemodialyzed patient are available.

It has been reported that some interleukins are generated during hemodialysis. It is known that activated monocytes during HD liberate certain mediators of immune response (Kim et al., 2011) therefore the study of individual mechanisms of immune response in patients may be very helpful in the process of individualized treatment. Insights into the current character of immune response can be achieved through the analysis of the methylation status of selected genes using existing arrays technologies. Such high throughput technologies will lead to the comparison of hemodialysis effects on the character of immune response in

DNA methylation patterns determined at the level of cfDNA in hemodialyzed patiens may

The clinical studies performed on hemodialyzed patients can also improve the theoretical knowledge about the clearance and turnover of circulating nucleic acids in connection to the

Today, there are various methodological approaches allowing the quantitative and qualitative characterization of cell-free nucleic acids in circulation of hemodialyzed patients. The study od cfDNA in plasma during the hemodialysis began 30 years ago and brought interesting results concerning the apoptotic origin of at least an important fraction of this DNA and increase of its plasma concentration during the hemodialysis session regardless of the used membranes and duration of session. In the past decade, the clearance of cfDNA from plasma was studied not only in hemodialyzed patients, and it was concluded, that it is very rapid (the half-life is given in minutes) and kidney does not play the major role in this process. In last years, the attention is paid to the examination of cell-free ribonucleic acids in the circulation with regard to their use as biomarkers under different pathological conditions. The application of such methodologies in the field of hemodialysis is still awaiting for the researchers, as well as the application of high throughput technologies likes Next Generation Sequencing and array technologies of different design. The connection of new technologies with clinical studies will result not only in clinical benefit but also in deeper insight into basic biological mechanisms of intracellular communication, epigenetic regulation and modulation of immune function at the individualized level leading to truly

This work was supported by the grant of Ministry of Education of the Czech Republic no.

**5. Future perspectives** 

**6. Conclusion** 

personalized medicine.

**7. Acknowledgment** 

MSM 0021620807.

Probably, such studies will be managed in near future.

different groups of patients with different comorbidities.

be predictive of response to specific drugs and drug combinations.

regulatory functions of this extremely interesting class of molecules.

disease (2 patients), primary amyloidosis (1 patient), renal artery stenosis (1 patient), renal transplantation (1patient), and unknown causes (3 patients).The study was designed to compare high-flux (n=37) vs. low-flux (n=15) and polysulfone (n=42) vs. modified cellulose (n=10) membranes, seven different membranes were used: Arylane H, Tricea 210, HF-80, Arylane M6, FX-60, FX-80, and P160. The effect of the dialysis length on cfDNA concentration after HD was evaluated.

Although correct interpretation of the results is difficult with regard to many variable factors, cfDNA levels increased more than four-fold in 75% of the patients after HD. The study by García-Moreira et al. (2006) has two priorities: 1/ the real-time PCR method was used for the first time to quantify cfDNA; 2/ clear data about the clearance of elevated cfDNA concentrations after HD were provided to scientific community. The authors reported a rapid decline to normal values within 30 minutes of completing a hemodialysis session.

In our study (Korabecna et al., 2008), we determined not only pre- and post-dialytic cfDNA concentrations in patients on hemodialysis (n=17) but we also examined non-dialyzed patients with chronic kidney disease (n=20) and patients on peritoneal dialysis (n=18) in comparison with healthy volunteers (n=20). We measured the total cfDNA levels using realtime PCR on GADPH gene. All patients involved in our study were hemodialyzed on highflux polysulfone membrane.

In HD patients, we found elevated postdialytic cfDNA values and reported high interindividual variability. We found also patients with lowered cfDNA values after hemodialysis. The levels of cfDNA in HD patients in pre-dialytic interval were significantly increased when compared with patients with chronic kidney disease and patients on peritoneal dialysis but there was no significant difference in comparison with healthy persons probably due to the very broad range of cfDNA concentrations obtained in our control group.

It seems that the study of quantitative and qualitative cfDNA parameters with regard to the process of hemodialysis can bring very interesting insights into the biological aspect of the treatment with important clinical consequences.

### **4.2 Cell-free nucleic acids in patients on peritoneal dialysis**

In our study mentioned above (Korabecna et al., 208), we use real-time PCR based on the GAPDH gene sequence amplification for quantification of cfDNA in plasma and overnight dialysate in patients on peritoneal dialysis. We determined the ratio of dialysate cfDNA/plasma cfDNA (P/D ratio) and discovered that this ratio inversely correlates with the duration of period for which the person is treated by peritoneal dialysis. The values of P/D ratio lower than one were found in patients dialysed for longer time (median 17 months).

Ozkaya et al. (2009) studied the plasma cfDNA in children on peritoneal dialysis and found significantly elevated values in comparison to healthy children and positive correlation with C- reactive protein levels in treated children.

Samples of peritoneal effluent with regard to their content of cfDNA were examined in the study by Pajek et al. (2009) to analyze the impact of PD solution with different biocompatibility and cytotoxic properties on the peritoneal membrane cells. Two PD solutions were tested: a conventional lactate buffered, acidic solution and a novel, bicarbonate/lactate buffered, neutral solution low in glucose degradation products. A significant decrease in appearance of cfDNA in effluent was observed with the novel PD solution.
