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NDT Advance Access originally published online on May 15, 2007
Nephrology Dialysis Transplantation 2007 22(9):2613-2622; doi:10.1093/ndt/gfm227
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Neutrophils in acidotic haemodialysed patients have lower intracellular pH and inflamed state

Joseph G. Wann1, Yung-Ho Hsu2, Chih-Ching Yang1,3,8, Chien-Sheng Lin4, Deborah W. Tai5, Jin-Shuen Chen6, Cheng-Wen Hsiao7 and Chau-Fong Chen1

1Department of Physiology, College of Medicine, National Taiwan University, 2Division of Nephrology, Department of Internal Medicine, Taipei Medical University-Wan Fang Hospital, 3Division of Nephrology, Taipei City Hospital-Heping Branch, 4Department of Emergency & Critical Care, Cheng Hsin Rehabilitation Medical Center, 5Division of Endocrinology and Metabolism, Department of Internal Medicine, Taipei Medical University-Wan Fang Hospital, 6Division of Nephrology, Department of Internal Medicine, 7Division of Colorectal Surgery, Department of Surgery, Tri-Service General Hospital, Taipei and 8Department of Internal Medicine, College of Medicine, National Yang-Ming University, University, Republic of China

Correspondence and offprint requests to: Dr Chau-Fong Chen, Department of Physiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan, China. Email: w7812{at}ms4.hinet.net



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The effect of intracellular pH (pHi) on neutrophils has not been clearly defined.

Methods. We used pre-dialysis neutrophils from three groups of haemodialysis (HD) patients having different levels of pre-dialysis plasma bicarbonate concentrations (PHCO3) and pH values (pre-dialysis PHCO3 of groups A, B and C were consistently ≤21, 21–26 and ≥26 mmol/l [mEq/l], respectively) and neutrophils from age- and sex-matched healthy controls to determine pHi, apoptosis, phagocytosis and oxidative burst reactions in vivo. We also studied, in group A, the effect of metabolic acidosis correction on neutrophil function. Furthermore, we investigated the effect of intracellular acidification on neutrophil functioning in vitro.

Results. Neutrophils from the HD patients in group A exhibited significantly lower pHi than those in groups B and C. In addition, group A neutrophils had significantly delayed apoptosis, enhanced phagocytosis and increased oxidative burst reactions compared with those in groups B and C. These alterations in neutrophil function in group A were reduced by correcting metabolic acidosis over a period of 1 month. Moreover, our in vitro studies demonstrated that the pHi of neutrophils is positively correlated with apoptosis and inversely correlated with phagocytosis and oxidative burst reactions.

Conclusion. HD patients having low PHCO3 exhibited low neutrophil pHi. This intracellular acidification may contribute to the delayed apoptosis, enhanced phagocytosis and increased oxidative burst reactions observed in these neutrophils compared with neutrophils having normal or higher pHi.

Keywords: apoptosis; haemodialysis; intracellular pH; neutrophil; phagocytosis



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
Low pre-dialysis plasma bicarbonate (Formula ) concentrations (PHCO3) are still commonly found in a number of haemodialysis (HD) patients, and even in subjects that are apparently well dialysed on standard bicarbonate dialysis (35 mmol/l) (mEq/l) [1,2]. Here, we studied uraemic patients on maintenance haemodialysis that had been divided into three groups of patients by three consecutive monthly pre-dialysis PHCO3 (≤21, 21–26, ≥26 mmol/l) [3]. These three groups of patients differed in terms of anthropometry, nutritional and biochemical markers, suggesting that they may also differ in physiological and/or patho-physiological status. Whether they exhibited any behavioural differences remains unknown.

Although uraemic acidosis is mild, long-term metabolic acidosis of this type is associated with many adverse effects, including negative nitrogen balance [4], increased protein decomposition [5], fatigue, bone lesion [6], hypercalciuria [7], enhancement of parathyroid hormone secretion [8], hyperphosphataemia, reduced effect of 1{alpha}-hydroxylase [9], impaired cardiovascular function (depression of myocardial contractility, sensitization to arrhythmia [10], increased pulmonary vascular resistance [11]), hormonal disturbances [12], insulin resistance [13], gluconeogenesis cycle disorders [14] and altered triglyceride metabolism [15]. However, little is known about the effect of uraemic acidosis on immune regulation in HD patients.

Neutrophils are relatively short-lived and they constitutively undergo apoptosis. Apoptotic neutrophils are phagocytosed by macrophages without release of pro-inflammatory mediators, leading to limitation of tissue injury and resolution of the inflammatory process. A suppressed neutrophil apoptosis results in prolongation of its life span and may be beneficial for the host defense against systemic bacterial invasion. However, prolonged survival of activated neutrophils could cause the uncontrolled release of cytotoxic metabolites and pro-inflammatory substances, and lead to amplified systemic inflammation, tissue injury and organ failure. Thus, a balance between neutrophil apoptosis and phagocytosis is considered to be important for HD patients, who are generally considered to be in an inflamed state.

Surprisingly, few investigators have studied the effects of extracellular pH (pHe), intracellular pH (pHi) or both on neutrophil function in HD patients. The present study was designed to evaluate in vivo neutrophil pHi and function, which included apoptosis, phagocytosis and oxidative burst reactions in three groups of HD patients with different pre-dialysis PHCO3 and pH values. We additionally investigated the in vitro effects of altered pHe and pHi (within the acidic pH range) on the regulation of neutrophil function. Finally, we studied the effect of correction of metabolic acidosis on neutrophil function in acidic-HD patients.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
In vitro study design
This study consisted of a multi-centred (3 HD centres), comparison of three groups of HD patients (group A had a pre-dialysis PHCO3 that was consistently ≤21 mmol/l and pH ≤7.38; group B had a pre-dialysis PHCO3 that was consistently between 21 and 26 mmol/l and pH 7.38–7.42; and group C had a pre-dialysis PHCO3 that was consistently ≥26 mmol/l and pH ≥7.42.) with a group of age-, sex-matched healthy subjects (group CN) in order to study neutrophil pHi and function, including apoptosis, phagocytosis and oxidative burst reactions. To evaluate the effect of metabolic acidosis on neutrophils, we investigated neutrophil functions in acidic HD patients (group A) after restoring their pre-dialysis PHCO3 to 23–26 mmol/l over a period of 1 month (group A+). To achieve this goal, the dialysate Formula concentration was increased from 35 mmol/l to 38–40 mmol/l. Blood was drawn from the arterial side of the arteriovenous (AV) fistula at the start of dialysis on a mid-week day to avoid different pre-dialysis PHCO3 levels on different weekdays. During blood sampling, the patient was at rest and there was no hand motion. Arterial blood gas was measured at 4°C from a whole blood sample taken anaerobically. The samples were analysed by an ABL 510 (Radiometer, Copenhagen, Denmark) within 5 min to avoid a decrease in PHCO3 due to delayed measurement. All measurements, except when mentioned otherwise, were performed within 1 h after the samples were taken. In addition, all measurements were assayed on at least three separate dialytic sessions for each patient and means of these data are presented.

In vitro study design
Neutrophils from each HD patient were assayed for neutrophil pHi and function, including apoptosis, phagocytosis and oxidative burst reactions, after incubating in different pH-adjusted (7.4, 6.5, 6.0, 5.5) culture media for 1 h. This created four groups of data (7.4, 6.5, 6.0, 5.5) for each patient. We chose bicarbonate-buffered RPMI 1640 supplemented with 1% FCS [FCS contained < 5 pg 100 ml of lipopolysaccaride (LPS) as certified by the manufacturer (HyClone, South Logan, UT, USA)], which has been previously adjusted to the desired pH values (7.4, 6.5, 6.0, 5.5). Aliquots of 106/ml were cultured at 37°C in humidified 5% CO2 (for cells suspended in medium at pH 7.4) or 7% CO2 (for cells suspended in medium at lower pH values) incubators to maintain the desired pH in media. The pH of the culture media did not significantly change during 1 h incubation (data not shown).

Selection of patients
After approval by the local medical ethics committee, 123 anuric patients undergoing maintenance HD (62 men and 61 women) gave informed consent and were entered the study. The mean age was 52.3 years (range, 40–74 years). Mean duration of dialysis was 39.6 months (range, 8–52 months). The underlying primary renal diseases were glomerulopathy (n = 58), polycystic kidney disease (n = 10), tubulointerstitial nephritis (n = 28), nephrosclerosis (n = 18) and unknown (n = 9). In addition, all individuals had serum C-reactive protein (CRP) levels below 0.6 mg/dl and none of them had diabetes mellitus, clinical infection, heart failure, liver cirrhosis or keto- and lactic-acidosis. Further, none of the individuals were taking calcium channel blockers, oral or intravenous active vitamin D or iron. The HD patients in groups A, B and C were screened for inclusion criteria in order to have similar [serum ferritin (200–250 µg/l), transferin saturation (TSAT) (20–50%), haemoglobin (10–11 g/dl), intact parathyroid hormone (i-PTH) [150–300 ng/l], length of HD (36–48 months) and Kt/V (1.2–1.5) (markers for adequacy of HD efficiency)] during the experimental time period (monthly intervals for 3 months). All patient laboratory values presented in this study were within inclusion limits. As shown in Table 1, there were no statistically significant differences in serum ferritin, TSAT, haemoglobin, i-PTH, length of HD or Kt/V in HD patients among groups A, B and C. The amount of maintenance erythropoietin supplement depended on clinical indications.


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  		Table 1. Subject characteristics in four study groups

 
Haemodialysis
HD was performed using a dialyser (Fresenius Polysulfone F8 HPS–F10 HPS) having a 1.8–2.4 m2 polysulfone membrane and a dialysate flow rate of 500 ml/min. The dialysate fluid composition was sodium 140 mmol/l, potassium 2 mmol/l, calcium 1.75 mmol/l (7.01 mg/dl), Formula 35 mmol/l, acetate 4 mmol/l and glucose 5.5 mmol/l (99.1 mg/dl). Treatment duration was 4 h and blood flow rates were around 200–250 ml/min. Anticoagulation was achieved using a loading dose followed by constant infusion of heparin. Net fluid removal was set on an individual basis according to the clinical needs of each patient.

Biochemical analysis
Albumin was measured with the Beckman Synchron CX9 Delta Chemistry Analyzer (GMI, Minnesota, USA) according to the manufacturer's instructions. Serum iron and total iron binding capacity were determined using spectrophotometry and detected by Integra 700/Roche Diagnostics (Basel, Switzerland). Ferritin was measured with a polyclonal goat anti-ferritin antibody labelled with acridinium ester and detected by ACS:180 (R) SE Automated CL System-Centaur (Bayer, Tarrytown, NY, USA). Intact-PTH was determined with biotinylated polyclonal goat anti-human PTH antibody compounded with acridinium ester-labelled polyclonal goat anti-human PTH antibody in phosphate buffer with goat IgG, and detected by the ADVIA Centaur Immunoassay system (Bayer, Tarrytown, NY, USA). Plasma CRP concentration was determined by rate nephelometry (Beckman Instruments, Inc., Galway, Ireland). Plasma IL-6 concentration was measured by ELISA (R&D Systems Inc. Minneapolis, MN, USA).

Neutrophil isolation and preparation
Heparinized blood was obtained from the AV fistula or graft in HD patients during pre-dialysis in groups A, B and C, and from age- and sex-matched healthy individuals who had taken no medication for at least 10 days before sampling. Neutrophils were isolated by sequential sedimentation in 6% of dextran in 0.9% sodium chloride for 40 min at 22°C. Centrifugation was in Ficoll-Paque at 1500 rpm for 30 min to pellet granulocytes and remaining erythrocytes. The resuspended pellet was centrifuged over an 81% isotonic Percoll gradient at 1750 rpm for 15 min to pellet erythrocytes. The diffused layer at the interface contained neutrophils which were harvested, washed, resuspended in a medium and counted. Cell viability was >98%, as determined by trypan blue exclusion. The preparation was routinely comprised of >95% neutrophils. Eosinophils were the principal contaminants as determined by Rapi-diff II staining (Diagnostic Developments, Lancashire, UK) of the cytocentrifuged samples.

Measurement of pHi
Neutrophils were isolated from the different groups as described earlier. Measurement of pHi was performed using carboxy-SNARF-1-AM as previously described [16]. Neutrophils [5 x 106/ml in indicated medium, previously pH adjusted for the in vivo studies, pH was adjusted to match the plasma pH in each study subject.] were loaded with 10 µM carboxy-SNARF-1-AM during 1 h at 37°C, washed in phosphate buffer solution (PBS), and resuspended in the same buffer at 5 x 106 in 100 µl. Assays were performed by flow cytometry, with excitation at 488 nm and emission analysis at FL2 and FL3. Ten thousand events were collected. The pHi was estimated from the ratio of emission intensities at the two wavelengths and was standardized by comparison with the fluorescence intensity ratios of cells whose pHi values were fixed by incubation with nigericin (10 µM) in high-potassium buffers, as previously described [16].

Neutrophil viability, apoptosis and necrosis
Flow cytometry
Cell apoptosis was measured by annexin V staining. Neutrophils were isolated from different conditions as described earlier and were washed in PBS, and density was adjusted to 1 x 106 ml [17]. Subsequently, 100 µl of the solution (1 x 105 cells) was transferred to a 5 ml culture tube. The 5 µl of annexin V-FITC (Pharmingen, San Diego, CA, USA) and 2 µl of propidium iodide (PI) were added and the cells were incubated for 15 min at room temperature in the dark and 400 µl of binding annexin V-buffer was added to each tube. The following controls were used: unstained cells, cells stained with Annexin V-FITC (no PI) and cells stained with PI (no Annexin V-FITC). The degree of apoptosis was assessed by flow cytometry (FACScal-Systems, San Diego, CA, USA) within 1 h. Cells staining positive for PI were considered dead cells (necrosis or late apoptosis), cells staining positive only for annexin V were considered apoptotic and cells negative for both were considered viable.

DNA fragmentation assay
Neutrophils were isolated from different conditions as described earlier and were lysed, and genomic DNA was extracted according to the protocol for apoptotic DNA laddering kit (R&D Systems). Samples (2 µg of DNA/lane) were analysed by gel electrophoresis (1.8% agarose) and ethidium bromide staining. The gel was visually examined under 305 nm of UV illumination.

Measurement of caspase-3 activity
Caspase-3 activity was measured with a colorimetric assay kit (Sigma). Neutrophils were isolated from different conditions as described earlier. After washing with PBS, neutrophils were lysed in 60 µl lysis buffer [50 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, pH 7.4, 5 mM 3-[(3-cholamindopropyl) dimethlyammonio]-1-propanesulfonate and 5 mM dithiothreitol], disrupted on ice by sonication and centrifuged (17 400 x g for 10 min). The supernatants (10 µl; 5 x 105 cell equivalents, containing ~20 µg protein) were incubated with a 2 mM acetyl-Asp-Glu-Val-Asp-p-nitroanilide substrate with or without 200 µM acetyl-Asp-Glu-Val-Asp-al, a specific caspase-3 inhibitor, at 37°C for 6 h in a total volume of 100 µl assay buffer. Caspase-3 activity was measured at 405 nm in a Model 3550-UV microplate reader (Bio-Rad), and expressed as nmol of p-nitroanilide liberated/106 cells/h [18]. The protein contents were determined with a BCA protein assay kit (Pierce).

Neutrophil phagocytosis
Bacteria phagocytosis capacity was assayed by means of a commercially available kit (PHAGOTEST; ORPEGEN Pharma, Heidelberg, FRG). For the in vitro study, neutrophils were isolated as described earlier and resuspended in 50% RPMI 1640, 50% autologous plasma in v/v (previously pH adjusted), then the opsonized FITC-labelled Escherichia coli were added and were incubated for 30 min in a humidified 5% or 7% CO2 incubators. For the in vivo study, heparinized whole blood was used for assay. The following procedure was performed following the manufacturer's instructions.

Oxidative burst reactions
Superoxide assay
The generation of superoxide (SO) anion was measured as the SO dismutase-inhibitable reduction of cytochrome-c by measuring absorbance at 550 nm as described by Babior et al. [19]. Neutrophils (106) were isolated as described above and were incubated in RPMI 1640 [previously pH adjusted (for the in vivo studies, pH was adjusted to match the plasma pH in each study subject)], with ferricytochrome-c (BDH Chemical, Toronto, Canada). Superoxide release was assayed with or without using phorbol myristate acetate (PMA; Sigma; final concentration, 0.5 µg/ml) as the stimulus. The reaction mixture was incubated at 37°C for 1 h in humidified 5% (for in vivo study) or 7% CO2 incubators and then centrifuged for 5 min at 800 x g at 4°C. Absorbance of the supernatant at 550 nm was read on a Beckman DU-8 spectrophotometer. The blank contained all the components listed earlier along with 60 µg of SO dismutase (Sigma) per millilitre to correct for ferricytochrome-c reduction by agents other than SO. The amount of reduced cytochrome-c present was calculated by using an extinction coefficient of 21.1/mM/cm at 550 nm. Results were expressed as nanomoles of SO/106 cells/1 h. Studies with PMA contained 60 mM ferricytochrome-c. At each of these concentrations, the total amount of ferricytochrome-c available for reduction, as determined by sodium dithionite, exceeded the amount reduced by PMA stimuli.

Hydrogen peroxide assay
Hydrogen peroxide release (H2O2) was measured by the oxidation of phenol red as described by Pick and Keisari [20]. Briefly, the reaction mixture (1.0 ml) consisted of RPMI 1640 [previously pH adjusted (for the in vivo studies, pH was adjusted to match the plasma pH in each study subject)] with 2 x 106 neutrophils, 0.28 mM phenol red (Sigma), 8.5 U of horseradish peroxidase (Worthington Diagnostics, Freehold, NJ), with or without PMA as the stimulus. After a 1 h incubation at 37°C in humidified 5% (was selected for in vivo study) or 7% CO2 incubators, 0.010 ml of 1M NaOH was added and the tubes were centrifuged at 800 x g for 5 min at 4°C. Absorbance of the supernatant at 610 nm was determined spectrophotometrically. Results were expressed as nanomoles of H2O2 produced per 106 neutrophils per 1 h.

Statistical analyses
All results are expressed as means ± SEM. Differences among groups were analysed by one-way analysis of variance (ANOVA) followed by Scheffe''s test for multiple comparisons. Pearson's correlation coefficient was used to determine the relationship between the neutrophil pHi and functions. A P-value of < 0.05 was considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Neutrophil apoptosis in patients and healthy individuals
In the initial experiments, flow cytometry using FITC-conjugated annexin V revealed that the neutrophils of the HD patients in groups A (A), B (B) and C (C) underwent rapid apoptosis. As shown in the A, B and C subparts of Figure 1A, 18–25% of the neutrophils were in the early stage of apoptosis with preserved membrane integrity and 1–4% were positive for both annexin V and PI. The neutrophils of healthy individuals (group CN) revealed a much lower degree of apoptosis. As shown in the CN subparts of Figure 1, approximately 9% of the neutrophils underwent apoptosis and <1% were positive for both annexin V and PI. As shown in Figure 1B–D, similar results were obtained in complementary experiments that were performed to assay DNA fragmentation. Flow cytometry and examination of caspase-3 activity revealed that neutrophil apoptosis was significantly increased in groups A, B and C compared with group CN. However, neutrophil apoptosis was significantly lower in group A than in groups B and C, and it did not differ between groups B and C. Furthermore, as shown in Figure 1A–D, we found that the lower neutrophil apoptosis in group A was increased after we restored the pHi by increasing the delivered bicarbonate dose (group A+). In accordance, we found that neutrophil apoptosis was significantly increased in the HD patients compared with the healthy individuals. In addition, neutrophil apoptosis in group A (that had lower PHCO3 and pHi values) was delayed compared with that in groups B and C. Moreover, the pHi-associated decrease in neutrophil apoptosis could be reversed by increasing the bicarbonate dose.


Figure 1
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  		Fig. 1. Neutrophil apoptosis was assessed by PI and annexin V-FITC staining in groups CN (CN; n=29), A (A; n = 35), B (B; n = 52), C (C; n = 36) and A+(A+; n = 35). (A) Representative results from flow cytometric analysis of binding of annexin V-FITC and PI in neutrophils from the five experimental groups. The relative distribution of cells manifesting viable (annexin– PI–), early (annexin+ PI–), and late (annexin+ PI+) apoptosis is shown as percentages. (B) Agarose gel electrophoresis of internucleosomal DNA fragmentation in neutrophils. The molecular markers are indicated to the left (M). Five experimental groups are indicated as CN, A, B, C and A+. (C) Results from statistical analysis showing the proportions of apoptotic cells (mean ± SEM) in the five experimental groups. *P < 0.01 compared with CN, #P < 0.01 compared with A. (D) Statistical results showed the caspase-3 activity (mean ± SEM) and expressed as nmol of p-nitroanilide liberated/106 cells/h in five experimental groups. *P < 0.01 compared with CN, #P < 0.01 compared with A.

 
Neutrophil phagocytosis in the patients and healthy individuals
Uptake of FITC-labelled bacteria by neutrophils in whole-blood samples was tested using a gram-negative species. Flow cytometric analysis revealed that neutrophil-opsonized phagocytosis occurred in groups A (A), B (B), C (C) and A+ (A+). As shown in the CN, B and C subparts of Figure 2A, 53–57% of the neutrophils phagocytosed FITC-labelled bacteria, while approximately 67% of the neutrophils from group A patients showed phagocytosis. As shown in Figure 2B, neutrophil phagocytosis was significantly increased in group A compared with groups CN, B and C. However, there was no significant difference in neutrophil-opsonized phagocytosis among groups B, C and CN. Furthermore, phagocytosis by neutrophils in group A+ was reduced. Similarly, neutrophil-opsonized phagocytosis was increased in HD patients having lower PHCO3 and pHi values compared with patients having normal or higher PHCO3 and pHi values. Again, this increase in neutrophil function observed in group A could be reduced by increasing the administered bicarbonate dose.


Figure 2
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  		Fig. 2. Neutrophil phagocytosis was assessed by uptake of FITC-labelled E. coli and DNA staining in groups CN (CN; n = 29), A (A; n = 35), B (B; n = 52), C (C; n = 36) and A+(A+; n = 35). (A) Representative results showing flow cytometric analysis of uptake of FITC-labelled E.Coli and DNA staining in neutrophils from the five experimental groups. The relative distribution of cells manifesting uptake of FITC-labelled E. coli is shown as percentages. (B) Results from statistical analysis showing the proportions of neutrophils that had phagocyted FITC-labelled E. coli (mean ± SEM) in the five experimental groups. *P < 0.05 compared with A.

 
Neutrophil-mediated oxidative burst reactions in the patients and healthy individuals
Oxidative burst reactions were estimated by SO and H2O2 production by neutrophils with or without PMA as a stimulus. There was no significant difference in baseline (resting) neutrophil SO or H2O2 production among the groups CN, A, B, C and A+ (data not shown). As shown in Figure 3A and B, PMA-stimulated SO and H2O2 production by the uraemic neutrophils from group A patients increased significantly compared with that of neutrophils from the individuals in groups CN, B and C. However, there was no significant difference in neutrophil SO and H2O2 production among groups CN, B, C and A+. Further, the incidence of neutrophil oxidative burst reactions in HD patients with normal pHi values was significantly lower in neutrophils from HD patients with lower pHi values, however, it was similar to that in neutrophils from healthy individuals. Furthermore, correction of metabolic acidosis restored the oxidative burst reactions in uraemic neutrophils.


Figure 3
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  		Fig. 3. Findings from statistical analysis showing (mean ± SEM) the production of superoxide (SO) (A) and of hydrogen peroxide (H2O2) (B) in uraemic neutrophils from groups CN (CN; n = 29), A (A; n = 35), B (B; n = 52), C (C; n = 36) and A+ (A+; n = 35). *P < 0.05 compared with A.

 
Effect of pHi on apoptosis and phagocytosis in uraemic neutrophils
To explore the role of pHi in the regulation of uraemic neutrophil function, neutrophils from the HD patients were incubated for 1 h in culture media having various pH values (7.4, 6.5, 6.0, 5.5), and were assayed for pHi as well as functions including apoptosis, phagocytosis and oxidative burst reactions. The pHi values of uraemic neutrophils after incubation for 1 h at various external pH levels are shown in Table 2. The pHi varied significantly with the external pH. As shown in Figure 4A and B, pHi was positively correlated with neutrophil apoptosis (A), whereas it was inversely correlated with phagocytosis (B). Accordingly, intracellular acidification of uraemic neutrophil delayed apoptosis but enhanced phagocytosis.


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  		Table 2. Uraemic neutrophil intracellular pH after 1 h incubation at various medium pH values

 

Figure 4
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  		Fig. 4. Correlations between neutrophil pHi and apoptosis (A) phagocytosis (B) SO, (C) H2O2 and (D) production from HD patients. Neutrophils (106 cells/ml) were incubated in RPMI 1640 at different pH (7.4, 6.5, 6.0, 5.5) for 1 h. After incubation, uraemic neutrophil pHi, apoptosis, opsonized phagocytosis, SO and H2O2 production were assayed as described in subjects and methods. For each experimental set-up n = 21.

 
Effect of pHi on oxidative burst reactions in uraemic neutrophils
Figure 4C and D illustrate the effects of pHi on SO and H2O2 production in neutrophils when PMA was used as a stimulus. The production of SO and H2O2 in neutrophils incubated in medium with a low pH increased significantly compared with that of neutrophils incubated in medium with a high pH. In addition, SO and H2O2 production in uraemic neutrophils was inversely correlated with pHi.



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
To the best of our knowledge, this is the first study to show that uraemic patients undergoing maintenance haemodialysis can be classified into three categories of PHCO3 and pH status and that these categories differ in terms of neutrophil function and pHi. The neutrophils from patients with low PHCO3 and pH values exhibited low pHi values. Further, the low pHi in this group may contribute to the delayed apoptosis, enhanced phagocytosis and increased oxidative burst reactions as compared with these processes in neutrophils having normal or higher pHi values. In addition, restoration of pre-dialysis PHCO3 and pH values in acidotic HD patients (group A) counteracted the alterations in neutrophil function and pHi. In vitro studies demonstrated that neutrophil pHi was positively correlated with apoptosis, but correlated inversely with phagocytosis and oxidative burst reactions. Finally, this is the first in vivo study to suggest that PHCO3 influences the regulation of pHi in circulating cells.

Uraemic neutrophil pHi and apoptosis
In vitro studies on neutrophils from uraemic patients have revealed that uraemic neutrophils undergo accelerated apoptosis [21]. However, little is known about the role that pHi plays in the regulation of uraemic neutrophil apoptosis. In this study, we observed that neutrophils from HD patients of group A, who have lower PHCO3 and pH, exhibited a lower pHi and delayed neutrophil apoptosis compared with those from patients in groups B and C. However, there was no significant difference in neutrophil apoptosis between groups B and C. In addition, and consistent with previous reports, neutrophils from the HD patients (groups A, B and C) underwent accelerated apoptosis compared with those from healthy individuals in group CN. Furthermore, in the in vitro study that was carried out in the acidic range of external pH, we demonstrated that pHi positively correlated with neutrophil apoptosis, although this correlation was not observed in the in vivo study. Our data suggest that intracellular acidification delayed uraemic neutrophil apoptosis. Nevertheless, further study is necessary to elucidate the relation between intracellular alkalification and uraemic neutrophil apoptosis.

During apoptosis, caspase are activated by cleavage of procaspases in a sequential manner. Caspase-3 is an effector caspase that plays a role in cell death induced by a variety of stimuli [22]. In this study, we observed that caspase-3 activity as well as the rate of uraemic neutrophil apoptosis was significantly higher in groups B and C than in groups A and CN. However, caspase-3 activity in uraemic neutrophils was not significantly correlated with apoptosis in the in vivo study (data not shown). Our data suggest that caspase-3 may be involved in the regulation of pHi-related neutrophil apoptosis in HD patients. Nevertheless, further study is necessary to elucidate the relations among caspase-3 activity, pHi and apoptosis in uraemic neutrophils.

Neutrophil pHi, phagocytosis and oxidative burst reactions
In previous work, there have been discrepancies in the effect of uraemia on neutrophil phagocytosis. A study from 1995 stated that, in general, the phagocyte activity is not impaired by uraemia [23]. This is inconsistent with earlier results reporting a reduced phagocytic activity in uraemic patients [24,25]. Recent studies suggested that neutrophil killing capability is reduced in HD patients, while the number of neutrophils that show phagocytic activity and produce reactive oxygen species (ROS) remains unchanged [26]. The mechanisms responsible for altered phagocytic activity of uraemic neutrophils are unclear and have been attributed to the time span from onset of dialysis [27], diabetes mellitus [28], iron stores [29], serum parathyroid hormone [30], dialyser bioincompatibility and reuse practice [31,32]. In vivo studies have demonstrated that pHi plays a role in the generation of SO radicals [33] as well as in chemotaxis [34] in normal neutrophils. However, the mechanisms by which pHi influences the regulation of uraemic neutrophil function have not been well established. Our present in vivo findings showed that uraemic neutrophils with low pHi values had enhanced phagocytosis and increased oxidative burst reactions in comparison to uraemic neutrophils having normal or high pHi values. In addition, our in vitro studies on uraemic neutrophils incubated in the acidic pH range demonstrated that pHi inversely correlated with phagocytosis and oxidative burst reactions. With regard to the generation of SO radicals, both our in vivo and in vitro data suggested that the production of ROS, including Formula and H2O2, showed increases under acidic conditions, resulting in the observed increase in cytotoxicity. These data are consistent with findings in normal neutrophils reported in previous studies. In contrast, Liberek and coworkers [35] suggested that a lowing of pHi in normal neutrophils was responsible for an observed inhibition of the respiratory burst reaction. On the other hand, our data do not necessarily contradict that of Trevani and co-workers [36] who used Formula -free buffer solutions in their experiments. They also demonstrated that the changes in pHi depended on the presence of extracellular Formula and they observed only a slight change in neutrophil pHi and function when HCl was added to the Formula -free medium. These findings suggest that Formula is necessary for the titration of extracellular H+, which in turn permits inward CO2 diffusion and subsequent intracellular acidification. In the present study, we observed that the HD patients in group A exhibited lower PHCO3 as well as reduced pHi values. Organic or inorganic acid in plasma, which cannot be efficiently removed by HD, can be titrated in the presence of Formula and this may result in intracellular acidification in uraemic neutrophils. Our data suggested that HD patients with low PHCO3 and pHi values, which indicate high acid production, may have radically altered neutrophil and/or immunological functions due to the low neutrophil pHi. Moreover, Formula may be one of the factors contributing to these discrepancies observed in neutrophil function. However, further study is necessary to explain these alterations.

Role of metabolic acidosis correction on uraemic neutrophil function
Pre-dialysis PHCO3 has been demonstrated to be negatively correlated with interdialytic weight gain and has been associated with ß2-microglobulin accumulation [37]. In addition, moderate pre-dialysis acidosis ranging from 20.1 to 21.0 mEq/l (mmol/l) appears to be associated with better nutritional status and lower relative risk for mortality or hospitalization than is observed in patients with normal ranges of mid-week pre-dialysis serum bicarbonate concentration (approximately 24 mEq/l) or severe acidosis (<16 mEq/l) [38]. Correction of metabolic acidosis by increasing delivered bicarbonate dose has been shown to decrease catabolism [39] and improve nutrition status [40]. In this study, we observed that alterations in neutrophil function in acidotic HD patients could be restored by correcting metabolic acidosis over a period of 1 month. Our data, therefore, suggest that the pHi-associated activation of neutrophil function in HD patients can be reduced by restoring neutrophil pHi and pHe. However, further studies will be necessary to elucidate the effects that metabolic acidosis correction has on the innate immune system. Although most patients exhibit some degree of post-HD alkalaemia, we did not evaluate post-HD neutrophil function since other factors (pHi, dialyser, dialysate, etc.) may have contributed to the alterations in neutrophil function.

In summary, we demonstrated that HD patients having low PHCO3 and pH values also exhibited low pHi. This intracellular acidification may contribute to the delayed apoptosis, enhanced phagocytosis and increased oxidative burst reactions observed in these neutrophils compared with neutrophils having normal or high pHi. In addition, these alterations in neutrophil functions in acidotic HD patients were restored by correcting metabolic acidosis over a period of 1 month. We propose that HD patients having a higher acid production may have an inflamed innate immune state and that PHCO3 may play an important role in the regulation of pHi in neutrophils. Therefore, more attention should be given to the role of PHCO3 as well as pHi in the immune regulation of uraemic neutrophils.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Movilli E, Zani R, Carli O, et al. Correction of metabolic acidosis increases serum albumin concentrations and decreases kinetically evaluated protein intake in haemodialysis patients: a prospective study. Nephrol Dial Transplant (1998) 13:1719–1722.[Abstract/Free Full Text]
  2. Gennari FJ. Acid-base balance in dialysis patients. Kidney Int (1985) 28:678–688.[Web of Science][Medline]
  3. Lin SH, Lin YF, Chin HM, Wu CC. Must metabolic acidosis be associated with malnutrition in haemodialysed patients? Nephrol Dial Transplant (2002) 17:2006–2010.[Abstract/Free Full Text]
  4. Hara Y, May RC, Kelly RA, Mitch WE. Acidosis, not azotemia, stimulates branched-chain, amino acid catabolism in uremic rats. Kidney Int (1987) 32:808–814.[Web of Science][Medline]
  5. Williams B, Layward E, Walls J. Skeletal muscle degradation and nitrogen wasting in rats with chronic metabolic acidosis. Clin Sci (1991) 80:457–462.[Web of Science][Medline]
  6. Lefebvre A, de Vernejoul MC, Gueris J, et al. Optimal correction of acidosis changes progression of dialysis osteodystrophy. Kidney Int (1989) 36:1112–1118.[Web of Science][Medline]
  7. Vora NM, Kukreja SC, York PA, et al. Effect of exercise on serum calcium and parathyroid hormone. J Clin Endocrinol Metab (1983) 57:1067–1069.[Abstract/Free Full Text]
  8. Lu KC, Lin SH, Yu FC, Chyr SH, Shieh SD. Influence of metabolic acidosis on serum 1,25(OH)2D3 levels in chronic renal failure. Miner Electrolyte Metab (1995) 21:398–402.[Web of Science][Medline]
  9. Reddy GS, Jones G, Kooh SW, Fraser D. Inhibition of 25-hydroxyvitamin D3–1-hydroxylase by chronic metabolic acidosis. Am J Physiol (1982) 243:E265–E271.[Web of Science][Medline]
  10. Marsiglia JC, Cingolani HE, Gonzalez NC. Relevance of beta receptor blockade to the negative inotropic effect induced by metabolic acidosis. Cardiovasc Res (1973) 7:336–343.[Web of Science][Medline]
  11. Brimioulle S, Lejeune P, Vachiery JL, et al. Effects of acidosis and alkalosis on hypoxic pulmonary vasoconstriction in dogs. Am J Physiol (1990) 258:H347–H353.[Web of Science][Medline]
  12. Schambelan M, Sebastian A, Katuna BA, Arteaga E. Adrenocortical hormone secretory response to chronic NH4Cl-induced metabolic acidosis. Am J Physiol (1987) 252:E454–E460.[Web of Science][Medline]
  13. DeFronzo RA, Beckles AD. Glucose intolerance following chronic metabolic acidosis in man. Am J Physiol (1979) 236:E328–E334.[Web of Science][Medline]
  14. Reaich D, Graham KA, Channon SM, et al. Insulin-mediated changes in PD and glucose uptake after correction of acidosis in humans with CRF. Am J Physiol (1995) 268:E121–E126.[Web of Science][Medline]
  15. Mak RH. Effect of metabolic acidosis on hyperlipidemia in uremia. Pediatr Nephrol (1999) 13:891–893.[CrossRef][Web of Science][Medline]
  16. Chow S, Hedley D, Tannock I. Flow cytometric calibration of intracellular pH measurements in viable cells using mixtures of weak acids and bases. Cytometry (1996) 24:360–367.[CrossRef][Web of Science][Medline]
  17. Martin-Malo A, Carracedo J, Ramirez R, et al. Effect of uremia and dialysis modality on mononuclear cell apoptosis. J Am Soc Nephrol (2000) 11:936–942.[Abstract/Free Full Text]
  18. Durand V, Renaudineau Y, Pers JO, Youinou P, Jamin C. Cross-linking of human FcgammaRIIIb induces the production of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor by polymorphonuclear neutrophils. J Immunol (2001) 167:3996–4007.[Abstract/Free Full Text]
  19. Babior BM, Kipnes RS, Curnutte JT. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest (1973) 52:741–744.[Web of Science][Medline]
  20. Pick E, Keisari Y. A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J Immunol Methods (1980) 38:161–170.[CrossRef][Web of Science][Medline]
  21. Cendoroglo M, Jaber BL, Balakrishnan VS, et al. Neutrophil apoptosis and dysfunction in uremia. J Am Soc Nephrol (1999) 10:93–100.[Abstract/Free Full Text]
  22. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science (1998) 281:1312–1316.[Abstract/Free Full Text]
  23. Ward RA, McLeish KR. Polymorphonuclear leukocyte oxidative burst is enhanced in patients with chronic renal insufficiency. J Am Soc Nephrol (1995) 5:1697–1702.[Abstract]
  24. Vanholder R, Ringoir S, Dhondt A, Hakim R. Phagocytosis in uremic and hemodialysis patients: a prospective and cross sectional study. Kidney Int (1991) 39:320–327.[Web of Science][Medline]
  25. Vanholder R, De Smet R, Jacobs V, et al. Uraemic toxic retention solutes depress polymorphonuclear response to phagocytosis. Nephrol Dial Transplant (1994) 9:1271–1278.[Abstract/Free Full Text]
  26. Anding K, Gross P, Rost JM, Allgaier D, Jacobs E. The influence of uraemia and haemodialysis on neutrophil phagocytosis and antimicrobial killing. Nephrol Dial Transplant (2003) 18:2067–2073.[Abstract/Free Full Text]
  27. Vanholder R, Van Biesen W, Ringoir S. Contributing factors to the inhibition of phagocytosis in hemodialyzed patients. Kidney Int (1993) 44:208–214.[Web of Science][Medline]
  28. Haag-Weber M, Cohen G, Horl WH. Clinical significance of granulocyte-inhibiting proteins. Nephrol Dial Transplant (2000) 15(Suppl 1):15–16.[Free Full Text]
  29. Waterlot Y, Cantinieaux B, Hariga-Muller C, et al. Impaired phagocytic activity of neutrophils in patients receiving haemodialysis: the critical role of iron overload. Br Med J (Clin Res Ed) (1985) 291:501–504.[Medline]
  30. Massry SG, Schaefer RM, Teschner M, et al. Effect of parathyroid hormone on elastase release from human polymorphonuclear leucocytes. Kidney Int (1989) 36:883–890.[Web of Science][Medline]
  31. Ward RA, Schmidt B, Blumenstein M, Gurland HJ. Evaluation of phagocytic cell function in an ex vivo model of hemodialysis. Kidney Int (1990) 37:776–782.[Web of Science][Medline]
  32. Rao M, Guo D, Jaber BL, et al. Dialyzer membrane type and reuse practice influence polymorphonuclear leukocyte function in hemodialysis patients. Kidney Int (2004) 65:682–691.[CrossRef][Web of Science][Medline]
  33. Simchowitz L. Intracellular pH modulates the generation of superoxide radicals by human neutrophils. J Clin Invest (1985) 76:1079–1089.[Web of Science][Medline]
  34. Simchowitz L, Cragoe EJ Jr. Regulation of human neutrophil chemotaxis by intracellular pH. J Biol Chem (1986) 261:6492–6500.[Abstract/Free Full Text]
  35. Liberek T, Topley N, Jorres A, et al. Peritoneal dialysis fluid inhibition of polymorphonuclear leukocyte respiratory burst activation is related to the lowering of intracellular pH. Nephron (1993) 65:260–265.[Web of Science][Medline]
  36. Trevani AS, Andonegui G, Giordano M, et al. Extracellular acidification induces human neutrophil activation. J Immunol (1999) 162:4849–4857.[Abstract/Free Full Text]
  37. Tzanatos H, Dalamangas A, Retsa K, Kapetanaki A, Agroyannis B. Relation of interdialytic water retention with apparent bicarbonate space, HCO3-, and pH in hemodialyzed uremic patients. Ren Fail (2005) 27:235–238.[Web of Science][Medline]
  38. Mehrotra R, Kopple JD, Wolfson M. Metabolic acidosis in maintenance dialysis patients: clinical considerations. Kidney Int Suppl (2003) 00:S13–S25.
  39. Bommer J, Locatelli F, Satayathum S, et al. Association of predialysis serum bicarbonate levels with risk of mortality and hospitalization in the Dialysis Outcomes and Practice Patterns Study (DOPPS). Am J Kidney Dis (2004) 44:661–671.[CrossRef][Web of Science][Medline]
  40. Blair D, Bigelow C, Sweet SJ. Nutritional effects of delivered bicarbonate dose in maintenance hemodialysis patients. J Ren Nutr (2003) 13:205–211.[CrossRef][Web of Science][Medline]
Received for publication: 24. 8.06
Accepted in revised form: 23. 3.07


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