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Erythrocyte caspase-3 levels in children with chronic kidney disease
Clinical Biochemistry 46 (2013) 219–224
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Erythrocyte caspase-3 levels in children with chronic kidney disease D. Polak-Jonkisz a,⁎, L. Purzyc b, M. Szcepańska c, I. Makulska a a b c
Department of Paediatric Nephrology, Wroclaw Medical University, ul. Borowska 213, 50-556 Wroclaw, Poland Department of Medical Biochemistry, Wroclaw Medical University, ul. Chalubinskiego 10, 50-368 Wroclaw, Poland Paediatric Dialysis Unit, Zabrze, Medical University of Silesia, ul. 3-go Maja 13-15, 41-800 Zabrze, Poland
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Article history: Received 28 July 2012 Received in revised form 27 September 2012 Accepted 15 October 2012 Available online 24 October 2012 Keywords: Erythrocyte Caspase-3 Lactate dehydrogenase Adenosine triphosphate Chronic kidney disease Children
a b s t r a c t Objectives: In chronic kidney disease (CKD), a number of intra- and extracellular factors, e.g., uremic toxins, mechanic, oxidative or osmotic stress — induce changes (rearrangements) in the structure of cytoplasmatic membrane, while also simultaneously deregulating blood cell metabolism and, in consequence, contributing to preliminary ageing and suicidal death of red blood cells (RBCs).The aim of the reported study was an evaluation of caspase-3 and lactate dehydrogenase activities and of ATP concentrations in erythrocytes as cellular responses to CKD progress. Design and methods: Conservatively treated sixty (60) CKD children were enrolled into the study and divided, according to CKD progression (stage I-IV). The control group consisted of twenty-five (25) healthy children. The activity of caspase-3 (Casp-3) and lactate dehydrogenase (LDH) were spectrophotometrically assayed in haemolysed erythrocytes. Adenosine triphosphate (ATPe) concentrations were measured by means of a luciferin-luciferase kit. Results: A gradual increase of LDH and ATP levels was observed in transition from CKD stage I to stage III. In Group IV, the levels of those parameters were statistically significantly lower than in the control group. The activity of Casp-3 in Group I was comparable to that in healthy children. The highest activity of Casp-3 was observed in Group III. Conclusions: 1. The activity of caspase-3 in RBCs of CKD children grows with progression of the disease. 2. The lower LDH activities and the ATP concentration drop below the values characteristic for the control group, as observed in stage IV of CKD, indicate a compromised energy balance. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction In chronic kidney disease (CKD), a number of extracellular, e.g., uremic toxins — as well as intracellular factors, such as ion transport disorders, together with mechanic, oxidative or osmotic stress — contribute to preliminary ageing and suicidal death of red blood cells (RBCs) [1–3]. The suicidal death of mature red blood cells demonstrates features typical for apoptosis, including cell shrinkage, membrane blebbing or exposure to phosphatidylserine on cell surface. But, when we take into account the lack of the main cellular matrix, which participates in the process (lack of nucleus, mitochondrium), the death of erythrocytes (eryptosis) takes actually a rather different course. The observations of RBC death-inducing mechanisms indicate a clear differentiation between the two (2) types of eryptosis: Ca2+-dependent and Ca2+-independent [4]. Numerous chemical and biological factors, which attack the cellular membrane, ligation of specific membranous autoantigens, growing stress events and/or energy depletion, are obvious eryptosis signalling steps [1]. Homeostasis of red blood cells requires continuous energy supplies, thus high enough adenosine triphosphate (ATP) concentrations and related ⁎ Corresponding author at: Department of Paediatric Nephrology, ul. Borowska 21-31, 50-556 Wroclaw, Poland. E-mail address: [email protected] (D. Polak-Jonkisz).
adenylate pool are necessary supporting factors [5]. ATP levels depend not only on its synthesis rate during either anaerobic glycolysis or reutilisation process from adenine and adenosine but also on the rate of its degradation and removal from the cell [6]. The glycolytic enzymes, form a complex at the inner surface of the RBC membrane and, particularly, with the protein, designated as the third protein band (band-3). These enzymes catalyse reactions, which are coupled together, where one enzyme takes the product of another enzyme as substrate. The activity of these enzymes (the flux rate of glycolysis) is dependent on the bond strength with cytoplasmic membrane [7]. According to many authors, it is the last stage of anaerobic glycolysis, catalysed by lactate dehydrogenase (LDH), which plays a particular role in the course of glycolyse [8–10]. In physiological conditions, the human system produces three types of polypeptide chains: muscular form (M-form), hepatic form (H-form) and X form, which is found only in the testes and sperm. These isoforms occur in cells in various proportions, forming homotetramers H4 and H4 and hybrid forms: H3M, H2M2,HM3. The activity of these isoforms and the vector of their impact depend on a number of factors, such as the sensitivity to pirogronate and lactate, pH of the environment and the interactions with lipid and protein parts of cell membranes. In the course of CKD, erythrocytes reveal disturbed calcium metabolism, leading to increased cytoplasmic calcium (Ca +2) levels in
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cells and to activation of calcium-dependent enzymes, such as calpains, caspases or transglutaminases [11,12]. In physiological conditions, these enzymes control the membrane structure and RBC cytoskeleton organisation. Oxidative and carbonyl stress affect proteins and erythrocyte membrane lipids, resulting in RBC injury, increased phosphatidylserine expression on cell surface and, subsequently, in an accelerated elimination of RBCs from circulation. On the other hand, oxidative stress activates caspase — an enzyme which, following our speculations, participates in protecting RBCs against premature haemolysis, while strengthening eryptosis . Caspase-aspartate-specific, cysteine-dependent proteases occur in the cell as inactive zymogens. Their activation consists in a specific proteolysis of the zymogen polypeptide chain. Two caspase types have been identified in RBCs: effector caspase-3 and initiating caspase-8, with an unclear mutual relationship between them. Out of many eryptosis supporting proteins, being substrates for caspase-3, we may, among others, distinguish: ATP-dependent calcium pump (PMCA — plasma membrane Ca2+ ATPase), calpains and band-3. These proteins participate in maintaining the membrane ion gradient and the “energy charge” level in cell [13–15]. In our earlier observations of children with CKD, decreased PMCA activities were observed in erythrocytes, together with a permanent drop of calmodulin concentration and with a simultaneous increase of cytoplasmic Ca +2 levels [11,12]. Caspase-3 participates in the network of glycolyse flux and PMCA activity controlling systems, thus it indirectly influences the energy levels of cells and cytoplasmatic Ca +2 concentrations. Therefore, it seemed fairly interesting to assess caspase-3 and LDH activities and assay ATP levels in red blood cells, as cellular responses to CKD progress. We assume that assaying these parameters may be particularly helpful for our efforts to learn the causes of shorter RBC life in the course of CKD.
Patients and methods Study population Sixty (60) children and adolescents with CKD (41 F/19 M) were enrolled into the study. They were divided by CKD progression and K/DOQI 2002 Guidelines [16]. Group I: 15 patients (age:11.2 ± 1.6 yr) with stage I CKD Group II: 15patients (age: 12.3 ±2.0 yr) with stage II CKD, Group III: 15 patients (age: 12.9 ± 1.2 yr), with stage III CKD and Group IV — 15 patients (age: 13.3 ±1.6 yr) with CKD at stage IV. Duration of the 1st stage CKD was 3.1–6.4 yr (mean 4.8 ± 1.4 yr); duration of the 2nd stage CKD was 0.9–5.8 yr (4.2 ± 1.3 yr); duration of the 3rd stage CKD was 2.3–6.9 yr (mean 4.9 ± 1.5 yr) and duration of the 4-th stage CKD was 2.1–5.2 yr (mean 3.9 ± 1.2 yr). The control group (C) consisted of 25 healthy, age-matched children (age: 12.6 ±1.5 yr) with normal kidney functions. The causes of CKD included: urinary tract malformations (28), hereditary glomerulopathy (14), chronic glomerulonephritis (11), the haemolytic uremic syndrome(4), unknown (3). Twenty-one (21) CKD patients (9 patients from Group III and 12 patients from Group IV) were treated with antihypertensive drugs (calcium channel blockers, angiotensin-converting enzyme inhibitors, beta-blockers), applied either in mono- or combined therapy. The other drugs, administered to the CKD patients, included: vitamin D3 analogues, vitamins C and B complex, folic acid, iron and ranitidine hydrochloride or omeprazole, while the patients did not need erythropoietin (EPO) administration. All the patients with CKD in stage III and IV were kept on phosphate-deficient diet and treated with compounds, stimulating intestinal phosphate binding. Dietary calcium intake was set at 500–600 mg of elementary calcium per day.
Laboratory measurements Morning blood samples were collected from the antecubital vein, in the sitting position of fasted children. Serum was separated after 1–3 hours from blood collection and the samples were stored at − 70 °C. The following parameters were determined in the serum: intact parathormone (iPTH), inorganic phosphate (Pi), total calcium (t-Ca), creatinine (cr). The methodology was described in detail in our previous study [11]. Selected laboratory parameters in the studied patients are shown in Figs. 1–3. The glomerular filtration rate (GFR) was calculated, according to the Schwartz formula [17]. The following parameters were assayed in red blood cells (RBC): caspase-3 (Casp-3), adenosine triphosphate (ATPe), lactate dehydrogenase (LDH). Preparation of erythrocytes (RBCs) A total of 2 mL of heparinised blood was centrifuged at 4 C (500 × g for 5 min). Plasma and buffy-coat were removed by suction and then RBCs were washed four times in an isotonic NaCl solution. Haemolysis was conducted by freezing at −20 °C and thawing four times. The obtained haemolysate of erythrocytes was stored at −20 °C and used after thawing. Haemoglobin was removed from erythrocyte haemolysates, according to the Loyevsky et al. method [18] , membranes were centrifuged and supernatant condensed and dialysed (Vivaspin 500) in the working buffer: 20 mM TRIS HCl (pH 7.4), 0.1 mM EGTA, 0.5 mM DTT and centrifuged at 4000×g. Lactate dehydrogenase (LDH) activity assay The activity of LDH was assayed by the modified method of Bergmeyer et al. [19], which employs spectrophotometric measurement of light absorbance drop (at wavelength of 340 nm) (corresponding to NADH concentration drop) at 37 °C. The applied substrates included 0.2 mM of NADH and 3 mM pirogronate in working buffer. The quantity of the enzyme, which catalyses transformation of 1 micromole of NADH during 1 minute in conversion to 1 mL of RBC, was accepted as the unit of LDH activity (IU). The intra- (during one day) and inter-assay (on different days) coefficients of variation for LDH at the quantification limit were b8.5 and b6.7%, respectively. Erythrocyte ATP concentrations (ATPe) Haemoglobin was removed from haemolysed RBC samples (100 μl of blood cells) and the remaining proteins were then denaturated by 5% HClO4 solution and centrifuged. The supernatant was subsequently activated with KHCO3 solution to pH 7.4, centrifuged were filtered (at 50.000 cut-off) prior to spectrophotometric measurements to remove haemoglobin residues. ATP concentration was assayed in the supernatant, using luciferin in a luciferase kit (Roche Diagnostic), following the manufacturer's recommendations. All those steps were performed at 4 °C and the results were expressed in μmol/LRBC. Caspase 3 (Casp-3) activity assay Erythrocytes (5% haematocrit) were prepared by being suspended in calpain inhibitors (1 mM of I and II inhibitors) and serine protease(1 mM of Ecotin)-containing working buffer. After centrifugation, haemolysis was induced by the freezing/thawing procedure, followed by repeated
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II stage
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500 450 400 350 300 250 200 150 100 50 0
Creatinine·10 3[ mg/dl ]
GRF [ ml/min/1,73m ]
Fig. 1. Mean values of serum creatinine and GFR in examined groups. Legends: cr: creatinine; GFR: glomerular filtration rate was calculated according to the Schwartz formula.
centrifugation (10,000 g×1 min.) and the obtained supernatant was used for protease activity assay. The enzyme activity was measured with an assay kit (Sigma-Aldrich), following the manufacturer's instructions. Acetyl-DEVD-pNA was used as colometric substrate. The enzyme quantity, which catalysed the formation of 1 pNA picomole during 1 minute, was regarded as the activity unit (IU). For Casp-3, the intra- and inter- assay %CV at the quantification limit were b 5.1 and b 3.1%, respectively.
informed consent to participate in the procedures. The investigation protocol was approved by the Bioethics Committee of Wroclaw Medical University and the study was conducted in accordance with the Helsinki Declaration.
Protein assay
ATPe (μmol/LRBC)
Protein concentrations were measured by the method of Bradford et al. with bovine albumin as standard [20].
Statistically significant ATP concentration rises were found in the studied children with CKD vs. the controls (p b 0.01). In turn, a gradual increase of ATPe concentration was observed in the groups of children with CKD: Group I (1570.53 ±178.88 μmol/LRBC), Group II (1694.33 ± 87.46 μmol/LRBC) and Group III (1902.47 ±135.09 μmol/LRBC). The lowest, statistically significant ATPe level was observed in the children of Group IV (1375,67± 66.62 μmol/LRBC) vs. either the healthy population or other groups of uremic patients, while the highest ATPe concentration was found in Group III — also statistically significantly higher in comparison with the other studied groups. The mean values of ATPe, concentration in red blood cells of the investigated groups are shown in Fig. 4.
Statistical analysis The obtained results of normal or log-normal distribution variables are expressed as mean values and standard deviations: x SD. The one-way variance analysis (ANOVA) and the post-hoc test (LSD test) were applied in order to compare means among the study groups. The comparisons between the study groups and the control were performed by Student's t-test. The relationships between variables were analysed by Pearson correlation coefficients. The differences were considered statistically significant at p b 0.05 level. Ethical considerations All the parents of the examined children, both patients and controls, as well as all the adolescents, enrolled into the study, gave their Control
I stage
Results Figs. 4–6 present the mean values of ATPe, Casp-3 and LDH in red blood cells of the investigated groups.
Casp-3 In the studied population of children with CKD, a statistically significant increase of caspase-3 activity was found, when compared with children in the control group (p b 0.001). The patients in Group I demonstrated the lowest caspase-3 activity (0.12 ± 0.04 IU) vs. the II stage
III stage
IV stage
12
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2
0 iPTH·10-1[ pg/ml ]
t-Ca [ mg/dl ]
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Fig. 2. Mean values of serum iPTH, total calcium (t-Ca) and inorganic phosphate (Pi) in examined groups. Legends: iPTH: intact parathormone; Pi: inorganic phosphate: t-Ca: total calcium.
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Fig. 3. Mean values of Hb and Ht in examined groups. Legends: Hb: hemoglobin; Ht : hematocrit.
other examined children with CKD, being also comparable with caspase-3 activity in the healthy children. In Group II, Casp-3 activity rose up to 0.24±0.05 IU. The highest caspase-3 activity characterised Group III (0.28±0.07 IU), the level statistically significantly differing from the values in Group I and in the control group (pb 0.001). The level of Casp-3 activity in Group IV was 0.24±0.06 IU, showing no statistically significant differences vs. either Group II or Group III. Fig. 5 presents the mean values of Casp-3 activity in red blood cells of the investigated groups. LDH (IU) Statistically significantly elevated LDH activity was one of the characteristic features in the studied population of children with CKD (0.41± 0.22 IU) vs. the controls (0.26 ± 0.07 IU) (p b 0.001). A gradual increase of LDH activity was found: Group I (0.35± 0.10 IU) , Group II (0.47± 0.12 IU) and Group III (0.67± 0.13 IU). The lowest activity was found in patients of group IV (0.14± 0.04 IU), while the highest one was observed in Group III, where it was significantly higher in comparison to the other studied groups. Fig. 6 shows the mean values of LDH activity in red blood cells of the investigated groups. Positive correlations were observed in the following pairs of parameters: ATPe and LDH (r=0.75; P>0.001), Casp-3 and LDH (r=0.26; p>0.05) in CKD children, while inverse correlations occurred between ATPe and Casp-3 (r=−0.60; p>0.05) in Group I. Discussion Renal failure progression disturbs cell homeostasis, not only damaging cytoplasmic membrane but disturbing the whole cell cycle as well. A proper control of erythrocyte homeostasis requires continuous energy supplies, to maintain its stable and appropriate levels. Energy depletion irreversibly leads to uncontrolled cell ageing and death. A number of researchers assume the type of cellular death (necrosis or eryptosis) to be, 3500
1500
NS
*** ** NS
**
II stage
III stage
IV stage
Fig. 5. The mean values of Casp-3 activity in the red blood cells of the investigated groups. Legends: NS — non-significant level, *p b 0,05, **p b 0,01 and ***p b 0,001.
among others, associated with red-ox system and intracellular ATP concentration changes [21–24]. Proper intracellular ATP concentrations and the balance between energy production and use are very important for the physiological “energy charge” to be maintained at a stable level between 0.85 and 0.95 [5]. Intracellular ATP levels and the “energy charge” of erythrocytes, determined not only by the synthesis rate of anaerobic glycolysis and the process of reutilisation (“salvage”) of adenylate nucleotides. Also, important is the rate of ATP degradation and removal from the cell, as well as other control processes [6,25]. The gradual increase of ATPe concentrations, observed in the study from CKD stage I to stage III was associated with the active course of anaerobic glycolysis. Following Cao Z. et al. and Cole RH et al., the occurrence of deoxyhaemoglobin, observed in the course of CKD plays an important role in anaerobic glycolysis [26,27]. Deoxyhaemoglobin, together with parallel nitrite-modified haemoglobin, acting with protein band-3, dissociates glycolytic enzymatic complexes from band-3. This process enhances glycolysis flux and the ATP synthesis, as observed in our study in CKD stages I-III. The processes maintain appropriate levels of the erythrocyte ATP pool, despite its growing ejection from cells, enhanced by hypoxy, which increases in the course of CKD progression [26,27]. The high enough levels of ATPe, observed in CKD patients of Groups I-III, effectively controlled the activity of ATP-dependent enzymes, simultaneously preventing any changes in RBC cytoskeleton and cytoplasmic membrane structures [24]. In stage IV of the disease, an ATP drop was observed (1375.67 ± 66.62 μmol/LRBC), even below the control group level (1485.52 ± 206,38 μmol/LRBC). The decreased LDH activity (a parameter also evaluated in the our study) with simultaneous glycolysis flux suppression may have been the cause of reduced ATPe concentration in the children with advanced (stage IV) CKD. The observed erythrocyte ATP concentration level at 1375,67± 66.62 μmol/LRBC resulted from the “redirection” of glucose catabolism onto the pentose phosphate pathway (PPP). Subsequently,
LDH [ molNAD+/(min*mlRBC) ]
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0 children with CKD
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Fig. 4. The mean values of ATPe concentrations in the red blood cells of the groups investigated. Legends: NS — non-significant level, *p b 0,05, **p b 0,01 and ***p b 0,001.
children with CKD
Control
I stage
II stage
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IV stage
Fig. 6. The mean values of LDH activity in the red blood cells of the investigated groups. Legends: NS — non-significant level, *p b 0,05, **p b 0,01 and ***p b 0,001.
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NADPH + H+ (a metabolite which controls the balance in the glutathione red-ox system) was produced, along with 5-phophoribosyl-1pyraphosphate — a substrate for adenine phosphoribosyltransferase (ART-ase) — an enzyme, catalysing adenosine monophosphate (AMP) synthesis from adenine. Adenine permeates into RBC and synthesises AMP, doing it together with ART-ase (an enzyme, the activity of which is enhanced by metabolic acidosis, a condition observed in CKD) [6,28]. The other factors, which increase AMP concentrations (while, indirectly, also ATP), include hypoxia, elevated levels of 2,3- bisphosphoglycerate and calmodulin deficits, all of which are observed in the course of CKD [11,29]. These factors contribute to suppressed activity of AMP-deaminase, a key enzyme, participating in the catabolism of AMP in RBC. AMP-deaminase activity is accompanied by that of erythrocyte 5-nucleotidase, a bifunctional enzyme with nucleotidase/nucleoside phosphotransferase activity. An increased concentration of 2,3biphosphoglycerate enhances its transferase activity, leading to higher AMP concentrations and shifting the balance of the 2ADP = AMP +ATP reaction leftwards, towards ADP, i.e., a substrate necessary to support the flux of glycolysis and the ATP synthesis. The run-down of ATP in RBC not only leads to disturbances in intracellular homeostasis but it also compromises the biconcave shape of red blood cells [24,30]. One of the effects of a smaller erythrocyte ATP pool is erythrocyte transformation into echinocyte, which indirectly induces anaemia in renal failure. In the available literature, erythrocyte ATP was assayed only in adult patients with CKD in various clinical stages (conservative treatment, renal replacement therapy) [25,28,31]. In those observations, erythrocyte ATP concentrations were elevated in the patients with CKD, when compared to healthy subjects but, unfortunately, no particular stages of the disease were evaluated. In our study, all the children with CKD demonstrated statistically significantly increased levels of erythrocyte ATP vs. those in healthy children. However, the particular stages of CKD (from I to IV), revealed high dynamics of changes in the intracellular ATP levels. In the process of anaerobic glycolysis of erythrocyte, a particular role of lactate dehydrogenase is observed, the activity of which and the direction/vector of its effects being decisive for the rate of glycolysis flux and related energetic consequences and cellular red-ox [8,32]. Lactate dehydrogenase is an enzyme, catalysing the reversible pyruvate reduction to lactate with a simultaneous oxidation of reduced adenine dinucleotide (NADH). Among the five physiological isoforms of LDH, H4 is identified in erythrocytes, together with H3M, the latter in smaller volumes. Regarding RBC, a view has been accepted that LDH synthesises pyruvate, however, the course of action of the enzyme seems to depend on a number of factors, such as H and M subunit proportions, the NAD+/NADH and lactate/pyruvate concentration ratios and intracellular pH [9,33]. LDH binds with band-3 via GAPDH, forming an inactive complex. Dissociation of LDH and GAPDH molecules from band-3, enhances their enzymatic activity and — simultaneously — the mutual exchange between NAD+ and NADH two cofactors, necessary to maintain the activity of both enzymes [24]. A successive growth of LDH activity was observed in the studied population of children with CKD in stage I-III. The process maintained glycolysis at proper levels, what was — in turn — associated with the stable, high ATP concentrations in the studied uremic erythrocytes. It was also confirmed by the observed positive correlation between ATPe and LDH activities at significant levels (p b 0.001). Significant drops in LDH activity were presented by the children of Group IV in comparison with patients in the other groups and in the control group. It can be explained by calmodulin deficit in patients with CKD, which — regardless of high Ca+2 concentrations — limits glycolysis flux by suppressing calmodulin concentration-dependent enzymes [11]. In result, it leads to decreased concentrations of NADH and pyruvate — the substrates necessary for LDH activity. Moreover, in the opinion of many researchers, hypoxia, which accelerates the
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intracellular transport of lactates and H+ ions, may also contribute to LDH inactivation, which was the case with our patients in CKD stage IV [9]. Also, a significantly decreased activity of LDH and erythrocyte calmodulin deficits suppresses glycolysis flux and, consequently, the decrease of erythrocyte ATP synthesis in the course of CKD. Following Xue Q et al., a lower enzyme activity is indicative of human RBC senescence [8]. In CKD, the activation of caspases is determined by a number of exogenous and endogenous factors. In the opinion of many researchers, changes in the oxidative status of erythrocytes, together with activity of caspases, suggest erypthosis to be, in part, related to shorter lifespan of circulating RBC and to anaemia [1,34,35]. In the studied population of children with CKD, a gradual increase of caspase-3 activity was observed in stages I to III of the disease, with highest difference between Group I (0.12±0.04 IU) and Group II (0.24±0.05 IU), amounting to almost 100% of its base value. The observed activation of caspase-3 in stage II, III and IV of CKD, was indirectly associated with RBC protection against excessively high, toxic concentrations of intraerythrocyte calcium. High Ca+2 concentrations are characteristic for the process of renal failure progression [8,9,36]. Cytoplasmic calcium concentrations depend on its influx /efflux balance via the cytoplasmic membrane. PMCA (plasma membrane Ca2+ATPase) and its endogenous modulators play an important role in calcium efflux. Our earlier observations of erythrocytes in children with CKD revealed a drop in the activity of the pump with calmodulin deficit [8]. In PMCA, caspase cuts off both the cytoplasmic autoinhibitor domain and the calmodulin binding domain, leaving a shortened, but still active, structure of PMCA. The process of specific cleavage of PMCA by caspase-3 is of particular importance for cases of calmodulin deficit [35]. Decreased activities of caspase-3 in erythrocytes were observed in stage IV of renal failure in the studied population, in comparison with the children with either stage II (statistically insignificant) or stage III (statistically significant) of the disease. Nevertheless, the enzyme activity was still statistically significantly higher (p b 0.001) than in the group of healthy children, what ensures a proper functionality of PMCA. Thus maintaining the efflux of calcium ions from the erythrocyte. Therefore, one may think that caspase-3 participates in the protein–lipid network, controlling PMCA activity and, indirectly, in the proper control of glycolysis of calcium-dependent enzymes to maintain their correct activity levels. The increasing Casp-3 activity induces a controlled proteolysis of band-3, causing dissociation of the enzymatic complexes of glycolysis and enhancing their activity, including LDH. We speculate, that a positive correlation between Casp-3 and LDH, observed in our CKD children, confirms glycolysis flux (and ATP synthesis) in the attempt to stabilise the energy level of RBC. The increased activity of caspase-3 in all the stages of CKD also exerts certain effects on the RBC lipid-protein membrane structure. In experimental models of oxidant-treated RBCs, several independent groups have demonstrated a significant caspase-3 processing and catalytic activity, which is correlated with PS externalisation, erythrophagocytosis and with membrane changes, leading to band-3 aggregation and subsequent phagocytosis [37–40]. Following Mandal et al., phosphatidylserine externalisation process control in RBC by caspase-3 may take two courses: via direct injury of phospholipid translocase or, what is more probable, by an indirect influence of the controlling factors of flippases [37]. We may speculate that the role of caspase-3 in the development of uremic anaemia is fairly significant. However, not only this protease is responsible for phosphatidylserine translocation to the superficial monolayer of erythrocyte membrane and, in consequence, to the elimination of such changed red blood cells from circulation. Conclusions Our observations allow to conclude that anaerobic glycolysis plays a key role in the control of elevated ATP concentrations — as one of
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uremic erythrocyte energy level markers in the early stages of CKD. A significant fall of LDH activity in the pre-dialytic stage of CKD indirectly suppresses anaerobic glycolysis. In such a case, other cellular processes, e.g., a reutilisation of purine nucleotides, may prevail in the control of erythrocyte ATP concentrations. Caspase-3 activation and high ATP levels may be regarded as an effector mechanism of eryptosis, possibly contributing, along with other mechanisms, to shorter life span of uremic RBCs. Further research is needed in order to broaden our understanding of these complicated processes in uraemic erythrocytes. Conflict of interest statement All the authors declare no conflict of interests. References [1] Föller M, Huber SM, Lang F. Erythrocyte programmed cell death. Life 2008;60:661-8. [2] Cheung Wai W, Hoon Paik Kyung, Mak Robert H. Inflammation and cachexia in chronic kidney disease. Pediatr Nephrol 2010;25:711-24. [3] Eckardt KU. Pathophysiology of renal anemia. Clin Nephrol 2000;53:2-8. [4] Lui JC, Wong JW, Suen YK, Kwok TT, Fung KP, Kong SK. Cordycepin induced eryptosis in mouse erythrocytes through a Ca2+-dependent pathway without caspase-3 activation. Arch Toxicol 2007;81:859-65. [5] Ataullakhanov FI, Vitvisky VM. What determines the intracellular ATP concentration. Biosci Rep 2002;22:501-11. [6] Marlewski M, Smolenski RT. Incrased rate of adenine incorporation into adenine nucleotide pool in erythrocytes of patients with chronic renal failure. Nephron 2000;86:281-6. [7] Campanella ME, Chu H, Low PS. Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane. Proc Natl Acad Sci U S A 2005;102:2402-7. [8] Xue Q, Yeung ES. Variability of intracellular lactate dehydrogenase isoenzymes in single human erythrocytes. Anal Chem 1994;66:1175-8. [9] Terlecki G, Czapinska E. Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme. Cell Mol Biol Lett 2007;12:378-95. [10] Świderek K, Peneth K. Binding ligands and cofactor to L-lactate dehydrogenase from human skeletal and heart muscles. J Phys Chem 2011;115:6366-76. [11] Polak-Jonkisz D, Purzyc L, Laszki-Szczachor K, Musial K, Zwolinska D. The endogenous modulators of Ca2+-Mg2+-dependent ATPase in children with chronic kidney disease (CKD). Nephrol Dial Transplant 2010;25:438-44. [12] Polak-Jonkisz D, Purzyc L, Zwolinska D, Misial K. Ca2+-Mg2+-dependent ATP-ase activity and calcium homeostasis in children with chronic kidney disease. Pediatr Nephrol 2007;22:414-9. [13] Berg CP, Engels IH, Rothbart A, Lauber K, Renz A, Schlosser SF, et al. Human mature red blood cells express cspase-3 and caspase-8, but are devoid of mitochondrial regulators of apoptosis. Cell Death Differ 2001;8:1197-206. [14] Suzuki Y, Ohkubo N, Aoto M, Maeda N, Cicha I, Miki T, et al. Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties. Biorheology 2007;44:179-80. [15] Bratosin D, Tcacenco L, Sidoroff M, Cotoraci C, Slomianny C, Estaquier J, et al. Active caspase-8 and -9 in circulating human erythrocytes purified on immobilized annexin-V: a cytmetric demonstration. Cytometry A 2009;75:236-44. [16] K/DOQI. Clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis 2008;39:S1–S266.
[17] Schwartz GJ, Brion LP, Spitzer A. The use of plasma creatinine concentration for estimating glomerular filtration rate in infants, children and adolescents. Pediatr Clin North Am 1987;34:488-93. [18] Loyevsky M, LaVaute T, Allerson CR, Stearman R, Kassim OO, Cooperman S, et al. An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element. Blood 2001;98:2555-62. [19] Bergmeyer HU, Bernt E, Hess B. In: Bergmeyer HU, editor. Methods in Enzymatic Analysis. Veinheim: Verlag Chemie; 1965. p. 736-43. [20] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248-54. [21] Schaper J, Kostin S. Cell death and adenosine triphosphate. Circulation 2005;112:6-8. [22] Leist M, Single B, Castoldi AF, Kühnle S, Nicotera P. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 1997;185:1481-6. [23] Golstein P, Kroemer G. Cell death by necrosis: towards a molecular definition. Trends Biochem Sci 2007;32:37-43. [24] Betz T, Lenz M, Joanny JF, Sykes C. ATP-dependent mechanics of red blood cells. Proc Natl Acad Sci U S A 2009;106:15320-5. [25] Klarl BA, Lang PA, Kempe DS, Niemoeller OM, Akel A, Sobiesiak M, et al. Protein kinase C mediates erythrocyte programmed cell death" following glucose depletion. Am J Physiol Cell Physiol 2006;290:C244-53. [26] Cao Z, Bell JB, Mohanty JG, Nagababu E, Rifkind JM. Nitrite enhances RBC hypoxic ATP synthesis and relase of ATP onto vasculature: a new mechanism for nitrite-induced vasodilation. Am J Physiol Heart Circ Physiol 2009;297:H1494-503. [27] Cole RH, Malavalli A. Erythrocytic ATP release in the present of modified cell-free haemoglobin. Biophys Chem 2009;144:119-22. [28] Marlewski M, Smolenski RT, Szolkiewicz M, Aleksandrowicz Z, Rutkowski B, Swierczynski J. Accelerated degradation of adenine nucleotide in erythrocytes of patients with chronic renal failure. Mol Cell Biochem 2000;213:93-7. [29] Ogasawara Y, Funakoshi M, Ishii K. Glucose metabolism is accelerated by exposure to t-butylhydroperoxide during NADH consumption in human erythrocytes. Blood Cells Mol Dis 2008;41:237-43. [30] Park Y, Best CA, Auth T, Gov NS, Safran SA, Popescu G, et al. Metabolic remodeling of the human red blood cell membrane. Proc Natl Acad Sci U S A 2010;107: 1289-94. [31] Slominska EM, Szolkiewicz M, Smolenski RT, Rutkowski B, Swierczynski J. High plasma adenine concentration in chronic renal failure and its relation to erythrocyte ATP. Nephron 2002;91:286-91. [32] Gladden LB. Lactate metabolism: a new paradigm for the third millennium. J Physiol 2004;558:5–30. [33] Downer J, Sevinsky JR. Incorporating expression data in metabolic modeling: a case study of lactate dehydrogenase. J Theor Biol 2006;240:464-74. [34] Bratosin D, Tcacenco L, Sidoroff M, Cotoraci C, Slomianny C, Estaquier J, et al. Active caspase-8 and -9 in circulating human erythrocytes purified on immobilized annexin-V: A cytmetric demonstration. Cytometry A 2009;75:236-44. [35] Paszty K, Antalffy G. The caspase-3 cleavage product of the plasma membranę Ca2+-ATPase 4b is activated and appropriately targeted. Biochem J 2005;391:687-92. [36] Gafter U, Malachi T. Red blood cel calcium level in chronic renal failure. Effect of continuous ambulatory peritoneal dialysis. J Lab Clin Med 1990;116:386-92. [37] Mandal D, Prasun K. Caspase-3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes. FEBS Lett 2002;513:184-8. [38] Matarrese P, Straface E, Pietraforte D, Gambardella L, Vona R, Maccaglia A, et al. Peroxynitrite induces senescence and apoptosis of red blood cells through the activation of aspartyl and cysteinyl proteases. FASEB J 2005;19:416-8. [39] Miki Y, Tazawa T, Hirano K, Matsushima H, Kumamoto S, Hamasaki N, et al. Clearance of oxidized erythrocytes by macrophages: involvement of caspases in the generation of clearance signal at band 3 glycoprotein. Biochem Biophys Res Commun 2007;363:57-62. [40] Lang Florian, Qadri Syed M. Mechanisms and significance of eryptosis, the suicidal death of erythrocytes. Blood Purif 2012;33:125-30.
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Erythrocyte caspase-3 levels in children with chronic kidney disease D. Polak-Jonkisz a,⁎, L. Purzyc b, M. Szcepańska c, I. Makulska a a b c
Department of Paediatric Nephrology, Wroclaw Medical University, ul. Borowska 213, 50-556 Wroclaw, Poland Department of Medical Biochemistry, Wroclaw Medical University, ul. Chalubinskiego 10, 50-368 Wroclaw, Poland Paediatric Dialysis Unit, Zabrze, Medical University of Silesia, ul. 3-go Maja 13-15, 41-800 Zabrze, Poland
a r t i c l e
i n f o
Article history: Received 28 July 2012 Received in revised form 27 September 2012 Accepted 15 October 2012 Available online 24 October 2012 Keywords: Erythrocyte Caspase-3 Lactate dehydrogenase Adenosine triphosphate Chronic kidney disease Children
a b s t r a c t Objectives: In chronic kidney disease (CKD), a number of intra- and extracellular factors, e.g., uremic toxins, mechanic, oxidative or osmotic stress — induce changes (rearrangements) in the structure of cytoplasmatic membrane, while also simultaneously deregulating blood cell metabolism and, in consequence, contributing to preliminary ageing and suicidal death of red blood cells (RBCs).The aim of the reported study was an evaluation of caspase-3 and lactate dehydrogenase activities and of ATP concentrations in erythrocytes as cellular responses to CKD progress. Design and methods: Conservatively treated sixty (60) CKD children were enrolled into the study and divided, according to CKD progression (stage I-IV). The control group consisted of twenty-five (25) healthy children. The activity of caspase-3 (Casp-3) and lactate dehydrogenase (LDH) were spectrophotometrically assayed in haemolysed erythrocytes. Adenosine triphosphate (ATPe) concentrations were measured by means of a luciferin-luciferase kit. Results: A gradual increase of LDH and ATP levels was observed in transition from CKD stage I to stage III. In Group IV, the levels of those parameters were statistically significantly lower than in the control group. The activity of Casp-3 in Group I was comparable to that in healthy children. The highest activity of Casp-3 was observed in Group III. Conclusions: 1. The activity of caspase-3 in RBCs of CKD children grows with progression of the disease. 2. The lower LDH activities and the ATP concentration drop below the values characteristic for the control group, as observed in stage IV of CKD, indicate a compromised energy balance. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction In chronic kidney disease (CKD), a number of extracellular, e.g., uremic toxins — as well as intracellular factors, such as ion transport disorders, together with mechanic, oxidative or osmotic stress — contribute to preliminary ageing and suicidal death of red blood cells (RBCs) [1–3]. The suicidal death of mature red blood cells demonstrates features typical for apoptosis, including cell shrinkage, membrane blebbing or exposure to phosphatidylserine on cell surface. But, when we take into account the lack of the main cellular matrix, which participates in the process (lack of nucleus, mitochondrium), the death of erythrocytes (eryptosis) takes actually a rather different course. The observations of RBC death-inducing mechanisms indicate a clear differentiation between the two (2) types of eryptosis: Ca2+-dependent and Ca2+-independent [4]. Numerous chemical and biological factors, which attack the cellular membrane, ligation of specific membranous autoantigens, growing stress events and/or energy depletion, are obvious eryptosis signalling steps [1]. Homeostasis of red blood cells requires continuous energy supplies, thus high enough adenosine triphosphate (ATP) concentrations and related ⁎ Corresponding author at: Department of Paediatric Nephrology, ul. Borowska 21-31, 50-556 Wroclaw, Poland. E-mail address: [email protected] (D. Polak-Jonkisz).
adenylate pool are necessary supporting factors [5]. ATP levels depend not only on its synthesis rate during either anaerobic glycolysis or reutilisation process from adenine and adenosine but also on the rate of its degradation and removal from the cell [6]. The glycolytic enzymes, form a complex at the inner surface of the RBC membrane and, particularly, with the protein, designated as the third protein band (band-3). These enzymes catalyse reactions, which are coupled together, where one enzyme takes the product of another enzyme as substrate. The activity of these enzymes (the flux rate of glycolysis) is dependent on the bond strength with cytoplasmic membrane [7]. According to many authors, it is the last stage of anaerobic glycolysis, catalysed by lactate dehydrogenase (LDH), which plays a particular role in the course of glycolyse [8–10]. In physiological conditions, the human system produces three types of polypeptide chains: muscular form (M-form), hepatic form (H-form) and X form, which is found only in the testes and sperm. These isoforms occur in cells in various proportions, forming homotetramers H4 and H4 and hybrid forms: H3M, H2M2,HM3. The activity of these isoforms and the vector of their impact depend on a number of factors, such as the sensitivity to pirogronate and lactate, pH of the environment and the interactions with lipid and protein parts of cell membranes. In the course of CKD, erythrocytes reveal disturbed calcium metabolism, leading to increased cytoplasmic calcium (Ca +2) levels in
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.10.021
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cells and to activation of calcium-dependent enzymes, such as calpains, caspases or transglutaminases [11,12]. In physiological conditions, these enzymes control the membrane structure and RBC cytoskeleton organisation. Oxidative and carbonyl stress affect proteins and erythrocyte membrane lipids, resulting in RBC injury, increased phosphatidylserine expression on cell surface and, subsequently, in an accelerated elimination of RBCs from circulation. On the other hand, oxidative stress activates caspase — an enzyme which, following our speculations, participates in protecting RBCs against premature haemolysis, while strengthening eryptosis . Caspase-aspartate-specific, cysteine-dependent proteases occur in the cell as inactive zymogens. Their activation consists in a specific proteolysis of the zymogen polypeptide chain. Two caspase types have been identified in RBCs: effector caspase-3 and initiating caspase-8, with an unclear mutual relationship between them. Out of many eryptosis supporting proteins, being substrates for caspase-3, we may, among others, distinguish: ATP-dependent calcium pump (PMCA — plasma membrane Ca2+ ATPase), calpains and band-3. These proteins participate in maintaining the membrane ion gradient and the “energy charge” level in cell [13–15]. In our earlier observations of children with CKD, decreased PMCA activities were observed in erythrocytes, together with a permanent drop of calmodulin concentration and with a simultaneous increase of cytoplasmic Ca +2 levels [11,12]. Caspase-3 participates in the network of glycolyse flux and PMCA activity controlling systems, thus it indirectly influences the energy levels of cells and cytoplasmatic Ca +2 concentrations. Therefore, it seemed fairly interesting to assess caspase-3 and LDH activities and assay ATP levels in red blood cells, as cellular responses to CKD progress. We assume that assaying these parameters may be particularly helpful for our efforts to learn the causes of shorter RBC life in the course of CKD.
Patients and methods Study population Sixty (60) children and adolescents with CKD (41 F/19 M) were enrolled into the study. They were divided by CKD progression and K/DOQI 2002 Guidelines [16]. Group I: 15 patients (age:11.2 ± 1.6 yr) with stage I CKD Group II: 15patients (age: 12.3 ±2.0 yr) with stage II CKD, Group III: 15 patients (age: 12.9 ± 1.2 yr), with stage III CKD and Group IV — 15 patients (age: 13.3 ±1.6 yr) with CKD at stage IV. Duration of the 1st stage CKD was 3.1–6.4 yr (mean 4.8 ± 1.4 yr); duration of the 2nd stage CKD was 0.9–5.8 yr (4.2 ± 1.3 yr); duration of the 3rd stage CKD was 2.3–6.9 yr (mean 4.9 ± 1.5 yr) and duration of the 4-th stage CKD was 2.1–5.2 yr (mean 3.9 ± 1.2 yr). The control group (C) consisted of 25 healthy, age-matched children (age: 12.6 ±1.5 yr) with normal kidney functions. The causes of CKD included: urinary tract malformations (28), hereditary glomerulopathy (14), chronic glomerulonephritis (11), the haemolytic uremic syndrome(4), unknown (3). Twenty-one (21) CKD patients (9 patients from Group III and 12 patients from Group IV) were treated with antihypertensive drugs (calcium channel blockers, angiotensin-converting enzyme inhibitors, beta-blockers), applied either in mono- or combined therapy. The other drugs, administered to the CKD patients, included: vitamin D3 analogues, vitamins C and B complex, folic acid, iron and ranitidine hydrochloride or omeprazole, while the patients did not need erythropoietin (EPO) administration. All the patients with CKD in stage III and IV were kept on phosphate-deficient diet and treated with compounds, stimulating intestinal phosphate binding. Dietary calcium intake was set at 500–600 mg of elementary calcium per day.
Laboratory measurements Morning blood samples were collected from the antecubital vein, in the sitting position of fasted children. Serum was separated after 1–3 hours from blood collection and the samples were stored at − 70 °C. The following parameters were determined in the serum: intact parathormone (iPTH), inorganic phosphate (Pi), total calcium (t-Ca), creatinine (cr). The methodology was described in detail in our previous study [11]. Selected laboratory parameters in the studied patients are shown in Figs. 1–3. The glomerular filtration rate (GFR) was calculated, according to the Schwartz formula [17]. The following parameters were assayed in red blood cells (RBC): caspase-3 (Casp-3), adenosine triphosphate (ATPe), lactate dehydrogenase (LDH). Preparation of erythrocytes (RBCs) A total of 2 mL of heparinised blood was centrifuged at 4 C (500 × g for 5 min). Plasma and buffy-coat were removed by suction and then RBCs were washed four times in an isotonic NaCl solution. Haemolysis was conducted by freezing at −20 °C and thawing four times. The obtained haemolysate of erythrocytes was stored at −20 °C and used after thawing. Haemoglobin was removed from erythrocyte haemolysates, according to the Loyevsky et al. method [18] , membranes were centrifuged and supernatant condensed and dialysed (Vivaspin 500) in the working buffer: 20 mM TRIS HCl (pH 7.4), 0.1 mM EGTA, 0.5 mM DTT and centrifuged at 4000×g. Lactate dehydrogenase (LDH) activity assay The activity of LDH was assayed by the modified method of Bergmeyer et al. [19], which employs spectrophotometric measurement of light absorbance drop (at wavelength of 340 nm) (corresponding to NADH concentration drop) at 37 °C. The applied substrates included 0.2 mM of NADH and 3 mM pirogronate in working buffer. The quantity of the enzyme, which catalyses transformation of 1 micromole of NADH during 1 minute in conversion to 1 mL of RBC, was accepted as the unit of LDH activity (IU). The intra- (during one day) and inter-assay (on different days) coefficients of variation for LDH at the quantification limit were b8.5 and b6.7%, respectively. Erythrocyte ATP concentrations (ATPe) Haemoglobin was removed from haemolysed RBC samples (100 μl of blood cells) and the remaining proteins were then denaturated by 5% HClO4 solution and centrifuged. The supernatant was subsequently activated with KHCO3 solution to pH 7.4, centrifuged were filtered (at 50.000 cut-off) prior to spectrophotometric measurements to remove haemoglobin residues. ATP concentration was assayed in the supernatant, using luciferin in a luciferase kit (Roche Diagnostic), following the manufacturer's recommendations. All those steps were performed at 4 °C and the results were expressed in μmol/LRBC. Caspase 3 (Casp-3) activity assay Erythrocytes (5% haematocrit) were prepared by being suspended in calpain inhibitors (1 mM of I and II inhibitors) and serine protease(1 mM of Ecotin)-containing working buffer. After centrifugation, haemolysis was induced by the freezing/thawing procedure, followed by repeated
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500 450 400 350 300 250 200 150 100 50 0
Creatinine·10 3[ mg/dl ]
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Fig. 1. Mean values of serum creatinine and GFR in examined groups. Legends: cr: creatinine; GFR: glomerular filtration rate was calculated according to the Schwartz formula.
centrifugation (10,000 g×1 min.) and the obtained supernatant was used for protease activity assay. The enzyme activity was measured with an assay kit (Sigma-Aldrich), following the manufacturer's instructions. Acetyl-DEVD-pNA was used as colometric substrate. The enzyme quantity, which catalysed the formation of 1 pNA picomole during 1 minute, was regarded as the activity unit (IU). For Casp-3, the intra- and inter- assay %CV at the quantification limit were b 5.1 and b 3.1%, respectively.
informed consent to participate in the procedures. The investigation protocol was approved by the Bioethics Committee of Wroclaw Medical University and the study was conducted in accordance with the Helsinki Declaration.
Protein assay
ATPe (μmol/LRBC)
Protein concentrations were measured by the method of Bradford et al. with bovine albumin as standard [20].
Statistically significant ATP concentration rises were found in the studied children with CKD vs. the controls (p b 0.01). In turn, a gradual increase of ATPe concentration was observed in the groups of children with CKD: Group I (1570.53 ±178.88 μmol/LRBC), Group II (1694.33 ± 87.46 μmol/LRBC) and Group III (1902.47 ±135.09 μmol/LRBC). The lowest, statistically significant ATPe level was observed in the children of Group IV (1375,67± 66.62 μmol/LRBC) vs. either the healthy population or other groups of uremic patients, while the highest ATPe concentration was found in Group III — also statistically significantly higher in comparison with the other studied groups. The mean values of ATPe, concentration in red blood cells of the investigated groups are shown in Fig. 4.
Statistical analysis The obtained results of normal or log-normal distribution variables are expressed as mean values and standard deviations: x SD. The one-way variance analysis (ANOVA) and the post-hoc test (LSD test) were applied in order to compare means among the study groups. The comparisons between the study groups and the control were performed by Student's t-test. The relationships between variables were analysed by Pearson correlation coefficients. The differences were considered statistically significant at p b 0.05 level. Ethical considerations All the parents of the examined children, both patients and controls, as well as all the adolescents, enrolled into the study, gave their Control
I stage
Results Figs. 4–6 present the mean values of ATPe, Casp-3 and LDH in red blood cells of the investigated groups.
Casp-3 In the studied population of children with CKD, a statistically significant increase of caspase-3 activity was found, when compared with children in the control group (p b 0.001). The patients in Group I demonstrated the lowest caspase-3 activity (0.12 ± 0.04 IU) vs. the II stage
III stage
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0 iPTH·10-1[ pg/ml ]
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Fig. 2. Mean values of serum iPTH, total calcium (t-Ca) and inorganic phosphate (Pi) in examined groups. Legends: iPTH: intact parathormone; Pi: inorganic phosphate: t-Ca: total calcium.
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Fig. 3. Mean values of Hb and Ht in examined groups. Legends: Hb: hemoglobin; Ht : hematocrit.
other examined children with CKD, being also comparable with caspase-3 activity in the healthy children. In Group II, Casp-3 activity rose up to 0.24±0.05 IU. The highest caspase-3 activity characterised Group III (0.28±0.07 IU), the level statistically significantly differing from the values in Group I and in the control group (pb 0.001). The level of Casp-3 activity in Group IV was 0.24±0.06 IU, showing no statistically significant differences vs. either Group II or Group III. Fig. 5 presents the mean values of Casp-3 activity in red blood cells of the investigated groups. LDH (IU) Statistically significantly elevated LDH activity was one of the characteristic features in the studied population of children with CKD (0.41± 0.22 IU) vs. the controls (0.26 ± 0.07 IU) (p b 0.001). A gradual increase of LDH activity was found: Group I (0.35± 0.10 IU) , Group II (0.47± 0.12 IU) and Group III (0.67± 0.13 IU). The lowest activity was found in patients of group IV (0.14± 0.04 IU), while the highest one was observed in Group III, where it was significantly higher in comparison to the other studied groups. Fig. 6 shows the mean values of LDH activity in red blood cells of the investigated groups. Positive correlations were observed in the following pairs of parameters: ATPe and LDH (r=0.75; P>0.001), Casp-3 and LDH (r=0.26; p>0.05) in CKD children, while inverse correlations occurred between ATPe and Casp-3 (r=−0.60; p>0.05) in Group I. Discussion Renal failure progression disturbs cell homeostasis, not only damaging cytoplasmic membrane but disturbing the whole cell cycle as well. A proper control of erythrocyte homeostasis requires continuous energy supplies, to maintain its stable and appropriate levels. Energy depletion irreversibly leads to uncontrolled cell ageing and death. A number of researchers assume the type of cellular death (necrosis or eryptosis) to be, 3500
1500
NS
*** ** NS
**
II stage
III stage
IV stage
Fig. 5. The mean values of Casp-3 activity in the red blood cells of the investigated groups. Legends: NS — non-significant level, *p b 0,05, **p b 0,01 and ***p b 0,001.
among others, associated with red-ox system and intracellular ATP concentration changes [21–24]. Proper intracellular ATP concentrations and the balance between energy production and use are very important for the physiological “energy charge” to be maintained at a stable level between 0.85 and 0.95 [5]. Intracellular ATP levels and the “energy charge” of erythrocytes, determined not only by the synthesis rate of anaerobic glycolysis and the process of reutilisation (“salvage”) of adenylate nucleotides. Also, important is the rate of ATP degradation and removal from the cell, as well as other control processes [6,25]. The gradual increase of ATPe concentrations, observed in the study from CKD stage I to stage III was associated with the active course of anaerobic glycolysis. Following Cao Z. et al. and Cole RH et al., the occurrence of deoxyhaemoglobin, observed in the course of CKD plays an important role in anaerobic glycolysis [26,27]. Deoxyhaemoglobin, together with parallel nitrite-modified haemoglobin, acting with protein band-3, dissociates glycolytic enzymatic complexes from band-3. This process enhances glycolysis flux and the ATP synthesis, as observed in our study in CKD stages I-III. The processes maintain appropriate levels of the erythrocyte ATP pool, despite its growing ejection from cells, enhanced by hypoxy, which increases in the course of CKD progression [26,27]. The high enough levels of ATPe, observed in CKD patients of Groups I-III, effectively controlled the activity of ATP-dependent enzymes, simultaneously preventing any changes in RBC cytoskeleton and cytoplasmic membrane structures [24]. In stage IV of the disease, an ATP drop was observed (1375.67 ± 66.62 μmol/LRBC), even below the control group level (1485.52 ± 206,38 μmol/LRBC). The decreased LDH activity (a parameter also evaluated in the our study) with simultaneous glycolysis flux suppression may have been the cause of reduced ATPe concentration in the children with advanced (stage IV) CKD. The observed erythrocyte ATP concentration level at 1375,67± 66.62 μmol/LRBC resulted from the “redirection” of glucose catabolism onto the pentose phosphate pathway (PPP). Subsequently,
LDH [ molNAD+/(min*mlRBC) ]
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Fig. 4. The mean values of ATPe concentrations in the red blood cells of the groups investigated. Legends: NS — non-significant level, *p b 0,05, **p b 0,01 and ***p b 0,001.
children with CKD
Control
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Fig. 6. The mean values of LDH activity in the red blood cells of the investigated groups. Legends: NS — non-significant level, *p b 0,05, **p b 0,01 and ***p b 0,001.
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NADPH + H+ (a metabolite which controls the balance in the glutathione red-ox system) was produced, along with 5-phophoribosyl-1pyraphosphate — a substrate for adenine phosphoribosyltransferase (ART-ase) — an enzyme, catalysing adenosine monophosphate (AMP) synthesis from adenine. Adenine permeates into RBC and synthesises AMP, doing it together with ART-ase (an enzyme, the activity of which is enhanced by metabolic acidosis, a condition observed in CKD) [6,28]. The other factors, which increase AMP concentrations (while, indirectly, also ATP), include hypoxia, elevated levels of 2,3- bisphosphoglycerate and calmodulin deficits, all of which are observed in the course of CKD [11,29]. These factors contribute to suppressed activity of AMP-deaminase, a key enzyme, participating in the catabolism of AMP in RBC. AMP-deaminase activity is accompanied by that of erythrocyte 5-nucleotidase, a bifunctional enzyme with nucleotidase/nucleoside phosphotransferase activity. An increased concentration of 2,3biphosphoglycerate enhances its transferase activity, leading to higher AMP concentrations and shifting the balance of the 2ADP = AMP +ATP reaction leftwards, towards ADP, i.e., a substrate necessary to support the flux of glycolysis and the ATP synthesis. The run-down of ATP in RBC not only leads to disturbances in intracellular homeostasis but it also compromises the biconcave shape of red blood cells [24,30]. One of the effects of a smaller erythrocyte ATP pool is erythrocyte transformation into echinocyte, which indirectly induces anaemia in renal failure. In the available literature, erythrocyte ATP was assayed only in adult patients with CKD in various clinical stages (conservative treatment, renal replacement therapy) [25,28,31]. In those observations, erythrocyte ATP concentrations were elevated in the patients with CKD, when compared to healthy subjects but, unfortunately, no particular stages of the disease were evaluated. In our study, all the children with CKD demonstrated statistically significantly increased levels of erythrocyte ATP vs. those in healthy children. However, the particular stages of CKD (from I to IV), revealed high dynamics of changes in the intracellular ATP levels. In the process of anaerobic glycolysis of erythrocyte, a particular role of lactate dehydrogenase is observed, the activity of which and the direction/vector of its effects being decisive for the rate of glycolysis flux and related energetic consequences and cellular red-ox [8,32]. Lactate dehydrogenase is an enzyme, catalysing the reversible pyruvate reduction to lactate with a simultaneous oxidation of reduced adenine dinucleotide (NADH). Among the five physiological isoforms of LDH, H4 is identified in erythrocytes, together with H3M, the latter in smaller volumes. Regarding RBC, a view has been accepted that LDH synthesises pyruvate, however, the course of action of the enzyme seems to depend on a number of factors, such as H and M subunit proportions, the NAD+/NADH and lactate/pyruvate concentration ratios and intracellular pH [9,33]. LDH binds with band-3 via GAPDH, forming an inactive complex. Dissociation of LDH and GAPDH molecules from band-3, enhances their enzymatic activity and — simultaneously — the mutual exchange between NAD+ and NADH two cofactors, necessary to maintain the activity of both enzymes [24]. A successive growth of LDH activity was observed in the studied population of children with CKD in stage I-III. The process maintained glycolysis at proper levels, what was — in turn — associated with the stable, high ATP concentrations in the studied uremic erythrocytes. It was also confirmed by the observed positive correlation between ATPe and LDH activities at significant levels (p b 0.001). Significant drops in LDH activity were presented by the children of Group IV in comparison with patients in the other groups and in the control group. It can be explained by calmodulin deficit in patients with CKD, which — regardless of high Ca+2 concentrations — limits glycolysis flux by suppressing calmodulin concentration-dependent enzymes [11]. In result, it leads to decreased concentrations of NADH and pyruvate — the substrates necessary for LDH activity. Moreover, in the opinion of many researchers, hypoxia, which accelerates the
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intracellular transport of lactates and H+ ions, may also contribute to LDH inactivation, which was the case with our patients in CKD stage IV [9]. Also, a significantly decreased activity of LDH and erythrocyte calmodulin deficits suppresses glycolysis flux and, consequently, the decrease of erythrocyte ATP synthesis in the course of CKD. Following Xue Q et al., a lower enzyme activity is indicative of human RBC senescence [8]. In CKD, the activation of caspases is determined by a number of exogenous and endogenous factors. In the opinion of many researchers, changes in the oxidative status of erythrocytes, together with activity of caspases, suggest erypthosis to be, in part, related to shorter lifespan of circulating RBC and to anaemia [1,34,35]. In the studied population of children with CKD, a gradual increase of caspase-3 activity was observed in stages I to III of the disease, with highest difference between Group I (0.12±0.04 IU) and Group II (0.24±0.05 IU), amounting to almost 100% of its base value. The observed activation of caspase-3 in stage II, III and IV of CKD, was indirectly associated with RBC protection against excessively high, toxic concentrations of intraerythrocyte calcium. High Ca+2 concentrations are characteristic for the process of renal failure progression [8,9,36]. Cytoplasmic calcium concentrations depend on its influx /efflux balance via the cytoplasmic membrane. PMCA (plasma membrane Ca2+ATPase) and its endogenous modulators play an important role in calcium efflux. Our earlier observations of erythrocytes in children with CKD revealed a drop in the activity of the pump with calmodulin deficit [8]. In PMCA, caspase cuts off both the cytoplasmic autoinhibitor domain and the calmodulin binding domain, leaving a shortened, but still active, structure of PMCA. The process of specific cleavage of PMCA by caspase-3 is of particular importance for cases of calmodulin deficit [35]. Decreased activities of caspase-3 in erythrocytes were observed in stage IV of renal failure in the studied population, in comparison with the children with either stage II (statistically insignificant) or stage III (statistically significant) of the disease. Nevertheless, the enzyme activity was still statistically significantly higher (p b 0.001) than in the group of healthy children, what ensures a proper functionality of PMCA. Thus maintaining the efflux of calcium ions from the erythrocyte. Therefore, one may think that caspase-3 participates in the protein–lipid network, controlling PMCA activity and, indirectly, in the proper control of glycolysis of calcium-dependent enzymes to maintain their correct activity levels. The increasing Casp-3 activity induces a controlled proteolysis of band-3, causing dissociation of the enzymatic complexes of glycolysis and enhancing their activity, including LDH. We speculate, that a positive correlation between Casp-3 and LDH, observed in our CKD children, confirms glycolysis flux (and ATP synthesis) in the attempt to stabilise the energy level of RBC. The increased activity of caspase-3 in all the stages of CKD also exerts certain effects on the RBC lipid-protein membrane structure. In experimental models of oxidant-treated RBCs, several independent groups have demonstrated a significant caspase-3 processing and catalytic activity, which is correlated with PS externalisation, erythrophagocytosis and with membrane changes, leading to band-3 aggregation and subsequent phagocytosis [37–40]. Following Mandal et al., phosphatidylserine externalisation process control in RBC by caspase-3 may take two courses: via direct injury of phospholipid translocase or, what is more probable, by an indirect influence of the controlling factors of flippases [37]. We may speculate that the role of caspase-3 in the development of uremic anaemia is fairly significant. However, not only this protease is responsible for phosphatidylserine translocation to the superficial monolayer of erythrocyte membrane and, in consequence, to the elimination of such changed red blood cells from circulation. Conclusions Our observations allow to conclude that anaerobic glycolysis plays a key role in the control of elevated ATP concentrations — as one of
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uremic erythrocyte energy level markers in the early stages of CKD. A significant fall of LDH activity in the pre-dialytic stage of CKD indirectly suppresses anaerobic glycolysis. In such a case, other cellular processes, e.g., a reutilisation of purine nucleotides, may prevail in the control of erythrocyte ATP concentrations. Caspase-3 activation and high ATP levels may be regarded as an effector mechanism of eryptosis, possibly contributing, along with other mechanisms, to shorter life span of uremic RBCs. Further research is needed in order to broaden our understanding of these complicated processes in uraemic erythrocytes. Conflict of interest statement All the authors declare no conflict of interests. References [1] Föller M, Huber SM, Lang F. Erythrocyte programmed cell death. Life 2008;60:661-8. [2] Cheung Wai W, Hoon Paik Kyung, Mak Robert H. Inflammation and cachexia in chronic kidney disease. Pediatr Nephrol 2010;25:711-24. [3] Eckardt KU. Pathophysiology of renal anemia. Clin Nephrol 2000;53:2-8. [4] Lui JC, Wong JW, Suen YK, Kwok TT, Fung KP, Kong SK. Cordycepin induced eryptosis in mouse erythrocytes through a Ca2+-dependent pathway without caspase-3 activation. Arch Toxicol 2007;81:859-65. [5] Ataullakhanov FI, Vitvisky VM. What determines the intracellular ATP concentration. Biosci Rep 2002;22:501-11. [6] Marlewski M, Smolenski RT. Incrased rate of adenine incorporation into adenine nucleotide pool in erythrocytes of patients with chronic renal failure. Nephron 2000;86:281-6. [7] Campanella ME, Chu H, Low PS. Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane. Proc Natl Acad Sci U S A 2005;102:2402-7. [8] Xue Q, Yeung ES. Variability of intracellular lactate dehydrogenase isoenzymes in single human erythrocytes. Anal Chem 1994;66:1175-8. [9] Terlecki G, Czapinska E. Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme. Cell Mol Biol Lett 2007;12:378-95. [10] Świderek K, Peneth K. Binding ligands and cofactor to L-lactate dehydrogenase from human skeletal and heart muscles. J Phys Chem 2011;115:6366-76. [11] Polak-Jonkisz D, Purzyc L, Laszki-Szczachor K, Musial K, Zwolinska D. The endogenous modulators of Ca2+-Mg2+-dependent ATPase in children with chronic kidney disease (CKD). Nephrol Dial Transplant 2010;25:438-44. [12] Polak-Jonkisz D, Purzyc L, Zwolinska D, Misial K. Ca2+-Mg2+-dependent ATP-ase activity and calcium homeostasis in children with chronic kidney disease. Pediatr Nephrol 2007;22:414-9. [13] Berg CP, Engels IH, Rothbart A, Lauber K, Renz A, Schlosser SF, et al. Human mature red blood cells express cspase-3 and caspase-8, but are devoid of mitochondrial regulators of apoptosis. Cell Death Differ 2001;8:1197-206. [14] Suzuki Y, Ohkubo N, Aoto M, Maeda N, Cicha I, Miki T, et al. Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties. Biorheology 2007;44:179-80. [15] Bratosin D, Tcacenco L, Sidoroff M, Cotoraci C, Slomianny C, Estaquier J, et al. Active caspase-8 and -9 in circulating human erythrocytes purified on immobilized annexin-V: a cytmetric demonstration. Cytometry A 2009;75:236-44. [16] K/DOQI. Clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis 2008;39:S1–S266.
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