Introduction

Hemodiafiltration (HDF) enhances convective removal of middle- to high-molecular-weight uremic toxins. Several infusion modes (postdilution, predilution, mixed dilution, etc.) have been developed for HDF, and numerous studies have been carried out to investigate the solute removal efficiency of HDF under various infusion modes and operating conditions [1–9].

HDF or hemofiltration (HF) has been reported to yield better outcomes as compared to low-flux hemodialysis (HD), such as reduced mortality [10–12], lower frequency of intradialytic hypotension [12], and increased body mass index [12], indicating the supremacy of H(D)F over low-flux HD. Recently, Vilar et al. [13] compared postdilution on-line HDF with high-flux HD (a single-centre retrospective study), and showed a reduced mortality and lower frequency of intradialytic hypotension in the predominant on-line HDF group. Meanwhile, the predilution mode has also been reported to be advantageous by Locatelli et al. [14], based on the results of a prospective multicenter randomized study, which showed a lower frequency of intradialytic hypotension in the predilution HF and predilution HDF groups than in the low-flux HD group. The advantage of predilution HDF was also pointed out from the viewpoint of nutrition, including metabolism of amino acids and the kinetics of leptin [15, 16].

In the predilution mode, a large volume of diluted blood enters the dialyzer, therefore, the blood is less concentrated inside the dialyzer, which can decrease the cell–cell and cell–membrane contacts. Therefore, the predilution mode may result in less harmful effects on the blood cells during the blood circulation through the hollow fibers. To determine whether predilution HDF might indeed be associated with minimal damage to the blood cells, we compared predilution and postdilution HDF from the viewpoint of their effects on various blood cells, including neutrophils, lymphocytes and platelets.

Subjects and methods

Patients

The subjects were five stable hemodialysis patients (three men and two women) who were receiving treatment at Hashimoto Clinic, Kanagawa, Japan. Their mean age was 50.6 ± 10.0 years, and the mean duration of dialysis was 15.3 ± 8.3 years. The primary renal diseases were chronic glomerulonephritis (n = 3), polycystic kidney (n = 1), and vesicoureteral reflux (n = 1). The patients had no major comorbidities except one patient had suffered from dialysis-related amyloidosis. There are no other therapeutic managements that may affect the level of IL-6 or ICAM-1 during the period of the study. Drug administration during the study remained unchanged. The individual therapies of the patients before the start of the study were hemodialysis (HD) (n = 3) and predilution on-line HDF (n = 2). All protocols were approved by the Institutional Ethics Committee.

Study protocol

Firstly, all patients were subjected to predilution on-line HDF for 1 month. Then, postdilution on-line HDF was performed in Week 1, predilution on-line HDF was performed in Week 2 and HD was performed in Week 3. All patients were investigated during HDF or HD sessions on the last day of the week. The treatment conditions were as follows: HDF: 4 h at QB 200 mL/min and QD total 500 mL/min; amount of substituting fluid, 50 L per session for predilution and 10 L per session for postdilution HDF; HD: QB 200 mL/min and QD 500 mL/min. The FDY-180GW dialyzer (PEPA membrane; NIKKISO Co., Ltd., Tokyo, Japan) was used, and on-line HDF was performed using ultrapure dialysate as the substituting fluid. The study was performed between November and December 2008.

Measurements

Blood samples were collected from the sample port of a blood circuit just before the start and at the end of the treatment. Serum levels of interleukin (IL)-6 (high-sensitivity) and intercellular adhesion molecule (ICAM)-1 were measured by enzyme immunoassay. In addition, the phagocytotic and sterilizing functions of the neutrophils were also measured. The phagocytotic function of the neutrophils was evaluated by flow cytometry based on the amount of latex particles phagocytosed by the neutrophils. The sterilizing function of the neutrophils was evaluated by measuring the amount of reactive oxygen species produced during phagocytosis. Lymphocyte blastoid transformation [17] was evaluated by measurement of the [3H]-thymidine uptake. Lymphocyte blastoid transformation was induced by phytohemagglutinin (PHA; strongly activates CD4-positive cells) or concanavalin A (ConA; strongly activates CD8-positive cells), and the lymphocyte proliferative response was determined after incubation in the presence (stimulated incubation) or absence (non-stimulated incubation) of PHA/ConA. The stimulated index (SI), that is, the ratio of the lymphocyte proliferative response in the presence of mitogen to that in the absence of mitogen, was evaluated. Plasma levels of platelet-derived microparticles (PDMPs) were measured as a marker of platelet activation, by enzyme-linked immunosorbent assay. The concentrations of IL-6, ICAM-1 and PDMPs after dialysis were corrected using the hematocrit values to reflect the effects of concentration resulting from removal of water.

To evaluate the solute removal efficiency of HDF performed by the two dilution modes, the degree of removal of β2-microglobulin (β2-MG) and α1-microglobulin (α1-MG), and the amount of albumin loss were also examined. The concentrations of β2-MG and α1-MG after dialysis were corrected using the hematocrit values to reflect the effects of concentration resulting from removal of water.

During the study period, the endotoxin level and bacterial contamination of the dialysate were determined once a week using Toxinometer (MT-358; WAKO, Tokyo, Japan) and R2A media, respectively. The levels of endotoxin were below the limit of detection. Bacterial cultures of the dialysate were negative.

Statistical analysis

Wilcoxon’s matched pair signed-rank test was used for statistical analysis of the data, except for those on lymphocyte blastoid transformation. The results of lymphocyte blastoid transformation in response to mitogen stimulation were examined by a two-way ANOVA followed by the Bonferroni test. Probability values (P value) of less than 0.05 were considered to be statistically significant.

Results

The removal rate of β2-MG and α1-MG were approximately 75 and 40 %, respectively for both dilution modes (Fig. 1). These results indicate that this study was performed with high-efficiency HDF. There were no significant differences in the β2-MG or α1-MG removal rates between the two dilution modes. Albumin loss tended to be slightly higher in postdilution HDF, however, the difference was not significant.

Fig. 1
figure 1

β2-Microglobulin (β2-MG) and α1-microglobulin (α1-MG) removal rates and amount of albumin loss. There were no significant differences in the β2-MG or α1-MG removal rates and amount of albumin loss between predilution and postdilution hemodiafiltration (n = 5, mean + SD)

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In predilution HDF, the IL-6 level decreased significantly after treatment as compared with the value before treatment, whereas in the case of postdilution HDF, no significant change of the IL-6 level was observed after treatment as compared to the level before treatment. The degree of decrease of the ICAM-1 level was significantly greater after predilution HDF than after postdilution HDF (Fig. 2).

Fig. 2
figure 2

Changes in the levels of interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) after dialysis as compared with the levels prior to dialysis. Data are shown as the percent change relative to the pre-dialysis value (100 %). The IL-6 level decreased significantly after predilution hemodiafiltration (HDF). The ICAM-1 level was significantly lower after predilution HDF than after postdilution HDF (n = 5, mean + SD)

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Significant increase of the phagocytotic function of neutrophils was observed after predilution HDF, whereas a significant decrease of the phagocytotic function was observed after postdilution HDF. After HD, although the phagocytotic function of the neutrophils decreased, the change was not significant. No significant changes were observed in the sterilizing function of the neutrophils, as compared with the pretreatment findings, after any of predilution HDF, postdilution HDF or HD (Fig. 3).

Fig. 3
figure 3

Changes in the phagocytotic and sterilizing functions of neutrophils before and after dialysis. The phagocytotic function of neutrophils increased significantly after predilution hemodiafiltration (HDF) and decreased significantly after postdilution HDF. The sterilizing function of the neutrophils was maintained at almost 100 % after dialysis relative to that before dialysis, and no difference was observed between the dilution modes (n = 5, mean + SD)

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Comparison of the lymphocyte proliferative response in non-stimulated incubation before and after treatment revealed a tendency toward a decrease in the proliferative response after treatment in the predilution mode, whereas there was a tendency toward an increase in the response after treatment in the postdilution mode. The observed differences were, however, not significant (Fig. 4). In the case of HD, the values remained almost the same before and after treatment.

Fig. 4
figure 4

Changes in the lymphocyte proliferative response in non-stimulated incubation after dialysis. Data are shown by the percent change relative to the pre-dialysis value (100 %). The lymphocyte proliferative response after non-stimulated incubation was more pronounced after postdilution hemodiafiltration (HDF), but no significant difference was observed between the dilution modes (n = 5, mean + SD)

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The percent change in the SI for lymphocytic response to stimulation with PHA or ConA was significantly lower after treatment in the postdilution mode as compared with that after treatment in the predilution mode (Fig. 5). The percent change in the SI after treatment in the predilution mode was greater than 100 %, that is, the response to stimulation increased after the treatment, whereas after treatment in the postdilution mode, the percent change in the SI was less than 100 %, that is, the response to stimulation decreased after the treatment. For the case of HD, no significant difference in the percent change of the SI between the two dilution modes was observed.

Fig. 5
figure 5

Percent change in the stimulation index (SI) for lymphocytic response to phytohemagglutinin (PHA) and concanavalin A (ConA) stimulations after diagnosis. Data are shown by the percent change relative to the pre-dialysis value (100 %). Significant decrease in the responses to PHA and ConA stimulations was observed after postdilution hemodiafiltration (HDF) as compared with that after predilution HDF. In other words, predilution produces a better response to stimulation (n = 5, mean + SD). Two-way ANOVA followed by the Bonferroni test

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Significant increase of the PDMPs was observed after HDF as compared with the pre-treatment level for both dilution modes. However, there was no significant difference between the two dilution modes (Fig. 6).

Fig. 6
figure 6

Changes in the levels of platelet-derived microparticles (PDMPs) before and after dialysis. In both dilution modes, significant increase of the PDMPs was observed after dialysis. There was no significant difference between the dilution modes (n = 5, mean +SD)

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Discussion

In hemodialysis, contact of blood with a non-biological surface, such as a dialysis membrane, is unavoidable, and induces various biological responses. It is important to select the optimum dilution mode that would be associated with the smallest biological responses, in order to maximize the merits of HDF. In the present study, we compared predilution HDF (replacement fluid: 50 L per session) and postdilution HDF (replacement fluid: 10 L per session) in terms of their effects on the blood cells, including neutrophils, lymphocytes and platelets. The new findings of this study include: (1) A greater degree of decrease in the levels of IL-6 and ICAM-1 was observed after predilution HDF than after postdilution HDF; (2) the phagocytotic function of the neutrophils increased significantly after treatment in the predilution mode, whereas the phagocytotic function of the neutrophils decreased significantly after treatment in the postdilution mode; (3) the lymphocyte proliferative response to stimulation with PHA or ConA was significantly higher after treatment in the predilution mode than after treatment in the postdilution mode; (4) treatment in the two dilution modes had almost equivalent effect on platelet activation, in view of the changes in the PDMP level.

IL-6, which plays an important role in the induction of the inflammatory state in dialysis patients [18–21], decreased significantly after treatment using the predilution mode. The level of ICAM-1 also decreased significantly after predilution HDF than after postdilution HDF. ICAM-1 is known to be produced in the vascular endothelial cells upon cytokine stimulation. The level of ICAM-1 in dialysis patients is consistently higher than normal levels, and further increase is reported to be associated with a poor prognosis [22, 23]. IL-6 has a molecular weight of 21–28 kDa and ICAM-1 has a molecular weight of 75–115 kDa (in blood, ICAM-1 is present as a soluble form bound to other molecules and has a larger molecular weight). HDF has been reported to be superior to HD in terms of the ability for removal of proinflammatory cytokines [11, 24, 25]. Under the HDF conditions employed in the present study, there were no significant differences in the α1-MG removal rate or albumin loss, suggesting that the two dilution modes may have almost the same ability to remove IL-6 and ICAM-1. Our results showed that the concentrations of IL-6 and ICAM-1 were lower after treatment in the predilution mode than after treatment in the postdilution mode. Therefore, the production of these molecules is considered to be lower in HDF by the predilution mode. Nakamura et al. [25] also reported the level of pro-inflammatory cytokines after predilution or postdilution HDF for 5 patients. Increase in the serum levels of cytokines, IL-6, and ICAM-1, observed after postdilution HDF was suppressed by the predilution HDF, which were consistent with our results. In the present study, we also evaluated neutrophil phagocytotic function, lymphocyte function and platelet activation in addition to the cytokine levels.

The phagocytotic function of the neutrophils increased significantly after treatment in the predilution mode, but decreased after treatment in the postdilution mode, suggesting the advantage of predilution mode in terms of the effect on the phagocytotic function of the neutrophils. Neither of the dilution modes produced any decrease in the sterilizing function of the neutrophils. The lymphocyte proliferative response to stimulation with PHA or ConA was significantly lower after treatment in the postdilution mode as compared with that after treatment in the predilution mode. Furthermore, in the predilution mode, the response to stimulation increased after the treatment (percent change in SI greater than 100 %). These results suggest that the functions of the lymphocytes remain favorable in HDF performed using the predilution mode. The influence of the two dilution modes on platelet activation was considered to be almost the same, in view of the similar changes in the PDMP level. This may be attributable to platelet activation occurring at other sites, such as the blood pump, circulatory circuit, or air trap, or to the effects of dilution in the predilution mode being counteracted by the increase of the flow rate.

The findings of the present study suggest that in post-dilution HDF, due to the blood concentration inside the dialyzer, the cell–cell and cell–membrane contact becomes severe and triggers the blood cells, especially neutrophils and lymphocytes, to induce various biological responses. On the other hand, in predilution HDF, although the blood flow velocity inside the dialyzer is higher, the cell–cell and cell–membrane interactions are reduced because the blood is diluted before it enters the dialyzer.

We used IL-6 and ICAM-1 as markers of inflammation occurred during treatment. IL-6 is a well know pro-inflammatory cytokine which plays a direct important role in the induction of the inflammatory state. Inflammation is one of the important factors that exacerbate the poor prognosis for dialysis patients. Serum IL-6 level was significantly correlated with major parameters indicating malnutrition [19]. Moreover, higher IL-6 levels are associated with all-cause mortality without additional changes in the markers of nutritional status of the patients [21]. Circulating level of ICAM-1, which directly associated with inflammation, dyslipidaemia, and vascular events, increased in hemodialysis patients [22], ICAM-1 also become as predictors of long-term outcome in chronic hemodialysis patients [23]. In the present study, we selected these molecules as direct markers of inflammation but these markers are also indirectly related to the mortality of dialysis patients. Therefore predilution HDF, which could decrease the production of these molecules, is considered to have a strong advantage for dialysis patients. We have an experience that 1 month of predilution HDF reduced the symptom like itchiness, rest leg syndrome, and insomnia. Short period of predilution HDF to the patients with the symptom seems to be also effective. In the present study, excess cytokine production and leukocyte activation during postdilution HDF reduced when predilution HDF were carried out, so we think that predilution could be favored in dialysis patients even if we offer for some predetermined period of time.

There is a limitation that should be addressed regarding the present study. The number of patients was too small to generalize the results. Further studies are required to replicate our findings in a larger multicenter trial. This study was exploratory study. We intended to clarify the evaluation point we should be focusing on in the future study. From this study, leukocytes and inflammation is considered to be an important factor which was affected by dilution mode.

In summary, in a comparison of 50-L predilution HDF and 10-L postdilution HDF in this study, the predilution mode appeared to be a better dilution mode from the point of view of the effects on the blood cells, based on the results in respect of IL-6 production, ICAM-1 production, phagocytotic function of the neutrophils and lymphocyte blastoid transformation. According to the results of the present study, predilution HDF could be expected not only to efficiently eliminate middle- to high-molecular-weight uremic toxins, as shown in numerous previous studies, but also to improve the functions of neutrophils and lymphocytes. We conclude that predilution HDF could be more favorable for dialysis patients than postdilution HDF from the point of view of the effects on the blood cells, especially neutrophils and lymphocytes.