Which parameters affect cytosolic free calcium in polymorphonuclear leukocytes of haemodialysis patients?

István Kárpáti, Ildikó Seres, János Mátyus, Thomas Ben, György Paragh, Zsuzsa Varga and György Kakuk

First Department of Medicine, Medical and Health Centre, University of Debrecen, Debrecen, Hungary



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Cytosolic free calcium ([Ca2+]i) is an important second messenger during stimulation in a wide variety of cells, including polymorphonuclear leukocytes (PMNs). Its mobilization in PMNs is altered in various diseases such as atherosclerosis and ageing. In chronic haemodialysis (HD) patients, both atherosclerosis and accelerated ageing are well known. Therefore [Ca2+]i in resting PMNs of HD patients was determined along with certain parameters which might affect it, such as recombinant human erythropoietin (rHuEpo) treatment, calcium–phosphate balance, and biocompatibility of dialysis membranes.

Methods. PMNs were separated by density centrifugation and [Ca2+]i was determined by spectrofluorimetry using Quin 2/AM fluorescent dye. Laboratory parameters were determined by standard methods in clinical chemistry.

Results. It was found that [Ca2+]i in resting PMNs of HD patients not undergoing rHuEpo therapy was higher than that of controls. After 12-weeks of rHuEpo therapy, [Ca2+]i decreased to near normal level. The role of erythropoiesis in normalization of [Ca2+]i in resting PMNs was supported by PMN [Ca2+]i which was elevated in patients who had low haemoglobin (<100 g/l) or haematocrit (<0.30) values. In some patients, including those receiving rHuEpo treatment, [Ca2+]i remained high, suggesting a role for other parameters in increasing [Ca2+]i. One possible parameter might be the disturbed calcium–phosphate metabolism of chronic renal failure, because we found a strong correlation between [Ca2+]i and plasma iPTH levels in HD patients (r=0.743, P<0.001). [Ca2+]i was also elevated in PMNs of those patients who had either low plasma calcium or high plasma phosphate levels. PMN [Ca2+]i of HD patients correlated positively with the duration of HD (r=0.671, P<0.001). However, there was no correlation between [Ca2+]i and patient age. The dialysis procedure itself also transiently increased PMN [Ca2+]i HD patients, independently of the type of dialysis membrane.

Conclusion. PMN [Ca2+]i is modulated by various parameters in HD patients, including the degree of anaemia, disturbances of calcium metabolism, and duration of dialysis treatment. The elevated [Ca2+]i of resting PMNs might contribute to altered functions in these cells.

Keywords: cytosolic free calcium; erythropoietin; haemodialysis; iPTH; polymorphonuclear leukocytes



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Chronic renal failure (CRF) is associated with an increased risk of infectious complications, suggesting an impairment of cellular host defence. Several investigators reported altered polymorphonuclear leukocyte (PMN) functions in haemodialysis (HD) patients, such as altered generation of reactive oxygen radicals, impaired chemotaxis, phagocytosis, and bacterial killing [14]. In the initiation of these cellular reactions, mobilization of cytosolic free calcium as a second messenger plays an important role [5]. Increased iPTH secretion and altered calcium–phosphate metabolism may also contribute to impaired calcium homeostasis in various cells [6,7], including dysfunction in PMNs. Furthermore, erythropoietin (rHuEpo), the major regulator of erythropoiesis, was shown to change cytosolic free calcium ([Ca2+]i) in vitro in several cell types, including platelets [8], human BFU-E derived erythroblasts [9] and vascular smooth-muscle cells [10]. Recombinant human erythropoietin (rHuEpo) acts on [Ca2+]i through a pertussis toxin-sensitive, GTP-binding G protein in erythroblasts [11]. It was also demonstrated that after 3 months rHuEpo therapy, the originally elevated [Ca2+]i decreased in platelets of uraemic patients [12].

The efficacy of rHuEpo with respect to anaemia correction in patients on maintenance HD is well established [13,14]. An effect of rHuEpo on cell-mediated immunity was also demonstrated by enhanced T-cell response to mitogen stimulation [15] or by lymphocyte phenotyping [16]. However, there have been no studies on the effects of rHuEpo on PMN [Ca2+] in HD patients, although PMNs are known to play an important role in host defence reactions.

Therefore the present study was designed to determine [Ca2+]i concentrations of PMNs in HD patients, and to examine several parameters that might affect these levels, such as rHuEpo treatment, haemoglobin values before and after rHuEpo treatment, plasma iPTH, calcium–phosphate balance, dialysis membrane biocompatibility, time on HD, and age.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Effect of rHuEpo therapy
rHuEpo (Eprex, Janssen Cilag) was administered to five patients (mean age 30.4±4.6 years, mean time on dialysis 30±22 months, diagnosis four with glomerulonephritis and one with tubulointerstitial nephritis) three times weekly after the dialysis session, at a dose of 50–150 IU/kg body weight. The dose was increased gradually every week for a total of 12 weeks. Thereafter, rHuEpo administration was interrupted for another 8 weeks. PMN [Ca2+]i was determined before treatment, 8 and 12 weeks after rHuEpo treatment, and 8 weeks after the interruption of rHuEpo treatment.

Patients
[Ca2+]i was determined in PMNs of 20 chronic HD patients on rHuEpo therapy from the beginning of renal replacement treatment. The rHuEpo dose was variable, ranging between 30 and 230 IU/kg body weight, and depended on haematocrit and haemoglobin values (mean age 56±12 years, mean time on HD 23±13 months, eight with glomerulonephritis, four with tubulointerstitial nephritis, two with pyelonephritis, two with diabetes nephropathy, and four with end-stage renal disease of unknown origin). The patients were placed on HD therapy consisting of three 4-h sessions weekly, using 1.3 m2 polysulphone capillary dialyser (Hemoflow F60(S) High Flux, Fresenius, Fresenius AG, Hamburg, Germany) and bicarbonate dialysis solution containing 1.5 mmol/l calcium, except for the study examining the type of dialysis membrane (see below). The Kt/V ratio was 1.1±0.3. None of the patients in the study had evidence of liver disease, thyroid disorders or infectious diseases for 3 months prior to study. The patients received active vitamin D (calcitriol, Rocatrol, Roche, 0.5 µg every other day) and i.v. iron (Ferrlecit, ferri sorbit gluconate, Aventis Pharma, 62.5 mg every other week) supplementation at the end of dialysis session. Laboratory parameters are presented in Table 1Go.


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Table 1. Main laboratory parameters of the 20 chronic haemodialysis patients involved in the study

 
All patients gave informed consent, and the protocol was approved by the Ethical Committee of the University.

Effect of dialysis membrane on PMN [Ca2+]i
Ten patients from the above group (mean age 51±11 years, mean time on HD 19±10 months) were dialysed three times during 1 week using a high-flux polysulphone-type membrane (PS) (Hemoflow F60(S), Fresenius), and then three times during another week using a cuprophane-based membrane (Cup) (Hemoflow E3, UF 5.8, Fresenius). In both cases, a bicarbonate dialysis solution was used containing 1.5 mmol/l calcium. On the third day of dialysis with each of the different types of dialysis membrane, blood was taken before and after the dialysis session. PMNs were separated and [Ca2+]i was determined.

Effect of parathyroidectomy on PMN [Ca2+]i
One patient (age 39 years, time on HD 7.1 years, diagnosis pyelonephritis) had high levels of iPTH (56 pmol/l) and required parathyroidectomy. Before surgical intervention, the patient was given conventional treatment and received i.v. calcitriol (Calcijex, Abbott) 2–4 µg/dialysis session. PMN [Ca2+]i was determined before surgical intervention and 1 and 2 weeks after surgical intervention. PMNs were separated from fasting blood before dialysis.

Controls
PMN [Ca2+]i was determined in 15 age- and sex-matched control subjects. The controls had no documented liver, diabetes or infectious diseases, and no cardiovascular complications. All gave informed consent, and the protocol met with the rules of the Ethical Committee of the University.

Methods
Blood was withdrawn before dialysis and after an overnight fast. Samples for PMN separation were heparinized.

PMNs were separated by density centrifugation using Histopaque 1077 (Sigma, St Louis, Mo, USA) according to the method of Boyum [17]. Cell viability was approximately 92%, as determined by trypan blue exclusion.

Determination of [Ca2+]i was performed using Quin 2/AM (Sigma) fluorescent dye, from the method described by Tsien et al. [18] and as published elsewhere [19]. This method was used for [Ca2+]i determination because we assessed only resting levels of [Ca2+]i in PMNs. The [Ca2+]i in these experiments did not reach the mmol/l range, as was confirmed by diluted calcium standard and free Quin 2 dye.

Laboratory parameters were determined by standard methods of clinical chemistry.

Statistical analysis was performed using Spearman statistical analysis for determination of correlation coefficients and Wilcoxon tests for paired analysis. Graphics were drawn by Excel, version 5.0 (Microsoft, Richmond, USA).



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Effect of rHuEpo therapy
Table 2Go shows the changes in haematocrit values, haemoglobin levels, and other laboratory parameters before and during rHuEpo therapy, and 8 weeks after interruption of the therapy in the case of five patients who were dialysed for 3.74±2.3 years without rHuEpo treatment. Haematocrit and haemoglobin values increased progressively with time on rHuEpo, and reached a maximum after 8 weeks on rHuEpo therapy. The haemoglobin and haematocrit values returned to their original levels 8 weeks after the interruption of rHuEpo therapy. In parallel with the normalization of erythropoiesis, the originally elevated PMN [Ca2+]i of HD patients decreased after 8 weeks of rHuEpo. PMN [Ca2+]i decreased further during the following 4 weeks of rHuEpo therapy, and returned to its original high level after an 8-week interruption of the rHuEpo administration (Figure 1Go).


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Table 2. Changes in main laboratory parameters of five haemodialysis patients before and after rHuEpo therapy, as determined before and during rHuEpo therapy (8 and 12 weeks) and 8 weeks after the interruption of rHuEpo therapy

 


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Fig. 1. Effect of rHuEpo treatment on cytosolic free calcium in PMNs of HD patients, before receiving rHuEpo treatment, 8 and 12 week after receiving rHuEpo treatment, and 8 weeks after the interruption of rHuEpo treatment.

 
In HD patients receiving rHuEpo from the beginning of renal replacement treatment, there was no significant difference in mean concentrations of PMN [Ca2+]i compared with controls (102±32 nmol/l and 105±15 nmol/l respectively). However, PMN [Ca2+]i ranged between 51 and 244 nmol/l, suggesting that rHuEpo was not able to normalize it in all cases. This was confirmed by the fact that significant differences were found in PMN [Ca2+]i between patients with haemoglobin levels <100 g/l compared with those with concentrations >100 g/l (Figure 2AGo), and patients with haematocrits <0.30 compared with those with concentrations >0.30 (Figure 2BGo).



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Fig. 2. Cytosolic free calcium in PMNs of rHuEpo-treated HD patients. (A) Patients were divided by their haemoglobin levels (<100 g/l and >100 g/l). (B) HD patients were divided by their haematocrit values (<0.30 and >0.30).

 

Effect of dialysis membrane biocompatibility on PMN [Ca2+]i
PMN [Ca2+]i of rHuEpo-treated patients before dialysis was near normal, as mentioned above, and did not significantly differ between the two types of membrane used (PS 112±35 nmol/l vs Cup 108±42 nmol/l). During HD, PMN [Ca2+]i of HD patients increased independently of the type of dialysis membrane, and the elevation in [Ca2+]i at the end of dialysis was pronounced only when the Cup membrane was used (Figure 3Go).



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Fig. 3. Changes in cytosolic free calcium in PMNs of haemodialysis patients during treatment with polysulphone (PS) or cuprophane (Cup) dialysis membranes. Cytosolic free calcium was determined before and after dialysis.

 

Effect of calcium–phosphate metabolism on PMN [Ca2+]i of HD patients
PMN [Ca2+]i of HD patients was correlated with plasma iPTH (Figure 4Go). The role of iPTH in modulating [Ca2+]i in PMNs was further confirmed in one patient who needed parathyroidectomy because of severe secondary hyperparathyroidism. Before surgical intervention, when plasma iPTH level was 56 pmol/l, PMN [Ca2+]i was greatly elevated (264 nmol/l). Within 2 weeks after surgery, PMN [Ca2+]i returned to the normal range along with the fall in plasma iPTH (it was 112 nmol/l after 1 week, and 120 nmol/l after 2 weeks). Furthermore, significantly greater [Ca2+]i values were found in PMNs of those patients who had low plasma calcium (<2.1 mmol/l) or high phosphate levels (>1.45 mmol/l) than in those with normal plasma calcium or phosphate (Figure 5AGo and BGo respectively).



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Fig. 4. Correlation between cytosolic free calcium in PMNs of haemodialysis patients and plasma iPTH levels.

 


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Fig. 5. Effect of calcium–phosphate parameters on cytosolic free calcium in PMNs of HD patients. (A) Cytosolic free calcium in PMNs of HD patients with low (<2.1 mmol/l) and normal (>2.1 mmol/l) plasma calcium levels. (B) cytosolic free calcium in PMNs of HD patients with high (>1.45 mmol/l) and normal (<1.45 mmol/l) plasma phosphate levels.

 

Effect on [Ca2+]i of age and of time on maintenance HD
Resting PMN [Ca2+]i of HD patients did not correlate with age (r=0.12); however, it showed a significant and positive correlation (r=0.671, P<0.001) with the time spent on HD (Figure 6Go).



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Fig. 6. Correlation between cytosolic free calcium in PMNs of HD patients and the time on maintenance HD.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Intracellular free calcium plays an important role as a second messenger in the activation of a wide variety of cells. For this signalling mechanism to work, the resting concentration of calcium in the cytosol must be kept low. All parameters inducing a rise in the resting level of calcium in cells decrease their reactivity after stimulation [5,20,21].

rHuEpo, one of the main regulators of erythropoiesis, has been demonstrated to affect [Ca2+]i in several cell types [810,12]. In erythroblasts this occurred through a pertussis toxin-sensitive, GTP-binding G protein [11]. In a previous study the role of erythropoiesis in regulating [Ca2+]i was shown when a 3-month rHuEpo therapy study resulted in the decrease of originally elevated platelet [Ca2+]i in uraemic patients [12].

The results presented here demonstrate that rHuEpo affects cytosolic free calcium not only in erythropoietic cells (e.g. erythrocytes), but also in PMNs of HD patients. The long-term effect of rHuEpo to decrease [Ca2+]i in PMNs of HD patients is similar to its long-term effect on [Ca2+]i in the platelets of uraemic patients [12]. To decrease PMN [Ca2+]i, rHuEpo requires approximately 8 weeks of administration to be effective. The significantly elevated PMN [Ca2+]i values of HD patients with low haemoglobin (<100 g/l) and haematocrit values (<0.30), compared with patients with haemoglobin levels >100 g/l or haematocrit values >0.30 is in agreement with the concept that correction of anaemia improves PMN [Ca2+]i.

The role of rHuEpo in the normalization of PMN function is underscored by the findings that rHuEpo corrects superoxide anion production and phagocytosis in PMNs of HD patients [22,23]. Chen et al. [22] demonstrated that rHuEpo enhanced superoxide anion production in stimulated PMNs of HD patients when stimulation was induced through specific receptors such as chemotactic peptide receptors, but not when nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was stimulated directly. This could indicate that rHuEpo is beneficial when the signal transduction mechanism for the elevation of [Ca2+]i is involved (such as in the case of chemotactic peptide receptor). However, there is no effect when NADPH oxidase is stimulated directly, and this route of PMN activation is independent of mobilization of [Ca2+]i. Considering that elevated [Ca2+]i levels decrease the reactivity of PMNs, as was demonstrated during ageing [1921], a decrease in [Ca2+]i of resting cells necessarily improves cell reactivity after stimulation when [Ca2+]i is involved in the signal transduction mechanism.

However, according to our results in certain HD patients, including those receiving rHuEpo, PMN [Ca2+]i remained elevated, suggesting that other parameters than normalization of erythropoiesis affected PMN [Ca2+]i.

One of these parameters might be renal replacement therapy, because during HD, and independently of dialysis membrane bio(in)compatibility, a transient elevation in PMN [Ca2+]i of HD patients was observed. However, a significant rise in PMN [Ca2+]i was demonstrated when the Cup (bioincompatible) membrane was used for dialysis. Furthermore, PMN [Ca2+]i in HD patients correlated well with the time spent on maintenance HD. This suggests that impaired renal function might also contribute to altered PMN [Ca2+]i in HD patients.

Another parameter with documented importance in affecting [Ca2+]i in PMNs of HD patients was calcium–phosphate metabolism, which is regulated by iPTH. The close correlation between [Ca2+]i and plasma iPTH appears to confirm the important role of iPTH in this respect. In addition to plasma iPTH, plasma calcium and phosphate levels are also important: when these levels were normal, PMN [Ca2+]i was similar to that of healthy control subjects. Recently it was demonstrated that increases in dietary calcium induced reductions in platelet and erythrocyte [Ca2+]i, and decreases in plasma iPTH in healthy subjects [7], and that PTH-related peptide induced a slow and prolonged rise in [Ca2+]i in single porcine theca cells [24]. Furthermore, the correction of parathyroid dysfunction by surgical intervention normalized not only the plasma iPTH of one patient but also his PMN [Ca2+]i. This underscores the role of iPHT, calcium, and phosphate in the regulation of PMN [Ca2+]i.

In summary, the impaired host defence in patients with CRF on maintenance HD is due, at least partially, to pathological elevations of PMN [Ca2+]i. [Ca2+]i is modulated by rHuEpo, calcium metabolism (in particular iPTH, calcium, and phosphate) and transiently by HD with bioincompatible (Cup) membranes. Normalization of these parameters might improve immunological dysfunction in these patients.



   Notes
 
Correspondence and offprint requests to: Dr Zsuzsa Varga MSc PhD, 1st Department of Medicine, Medical and Health Centre, University of Debrecen, PO Box 19, H-4012 Debrecen, Hungary. Back



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
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Received for publication: 14. 3.00
Revision received 9.12.00.



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