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NDT Advance Access published online on June 7, 2007
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfm306
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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

Effects of NH4Cl-induced systemic metabolic acidosis on kidney mitochondrial coupling and calcium transport in rats

Leda Marcia A. Bento1, Marcia M. Fagian2, Anibal Eugênio Vercesi2 and José Antonio Rocha Gontijo1

1Laboratórios de Metabolismo Hidro-Salino e and 2Bioenergética, Departamentos de Clinica Médica e Patologia Clinica, Núcleo de Medicina e Cirurgia Experimental, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil

Correspondence and offprint requests to: J. A. R. Gontijo, Departamento de Clínica Médica, Faculdade de Ciências Medicas, Universidade Estadual de Campinas, 13083-100 Campinas, SP, Brazil. Email: gontijo{at}fcm.unicamp.br



   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. We have previously shown that chronic metabolic acidosis, induced in rats by NH4Cl feeding, leads to nephron hypertrophy and to a decreased water-salt reabsorption by the kidneys. Since mitochondria are the main source of metabolic energy that drives ion transport in kidney tubules, we examined energy-linked functions (respiration, electrochemical membrane potential and coupling between respiration and ADP phosphorylation) in mitochondria isolated from rat kidney and liver at 48 h after metabolic acidosis induced by NH4Cl.

Methods. Mitochondria isolated from the kidneys and liver of metabolic acidotic rats, induced by NH4Cl, was used to study of the oxygen consumption by Clark-type electrode, mitochondrial electrical transmembrane potential estimated by the safranine O method and the variations in free medium Ca2+ concentrations examined by absorbance spectrum of Arsenazo III set at the 675–685 nm wavelength pair.

Results Whole kidney and liver mitochondria isolated from 48 h acidotic rats presented higher resting respiration, lower respiratory control and a lower ADP/O ratio than controls. These differences in mitochondrial coupling, between respiration and oxidative phosphorylation (ATP synthesis), were totally corrected when experiments were carried out in the presence of carboxyatractyloside, GDP and BSA, indicating that mitochondrial uncoupling proteins are more active in acidotic rat kidneys. Interestingly, determination of Ca2+ transport demonstrated a faster rate of initial Ca2+ uptake by acidotic kidney mitochondria, which resulted in a lower concentration of extra-mitochondrial Ca2+ under steady-state conditions (Ca2+ set point) when compared with control mitochondria. In contrast, there were no significant differences in the rates of Na+ or ruthenium red induced Ca2+ efflux.

Conclusions. We suggest that the mild uncoupling and higher Ca2+ accumulation represents an adaptation of the mitochondria to cope with conditions of oxidative stress and high cytosolic Ca2+, which are associated with a decreased efficiency of oxidative phosphorylation that may explain, at least in part, the striking natriuresis observed under chronic acidosis. Finally, there were no changes in Ca2+ transport or coupling in liver mitochondria isolated from the acidotic rats.

Keywords: kidney mitochondria; NH4Cl-induced metabolic acidosis; oxidative phosphorylation; respiration; uncoupling



   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 References
 
There is a surprising lack of experimental data on the mechanisms of kidney dysfunction associated with chronic metabolic acidosis. In previous studies in rats [1,2] metabolic acidosis induced by NH4Cl resulted in an absolute increase in kidney mass associated with disturbances in renal sodium handling that were preceded by activation of kidney MAPK/ERKs signalling pathways. Under these acidotic conditions, the altered renal electrolyte handling may have resulted from a lack of energy availability for ion transport due to the high energy demand for renal anabolic processes [1].

Other studies have shown that ammonia cell toxicity is associated with alterations in mitochondrial bioenergetics [3], electrophysiological effects [4], disturbances in neurotransmitter function [5], glutamate-mediated cytotoxicity [6], oxidative stress [7] and is negatively correlated with gluconeogenesis activity and with renal sodium reabsorption [8]. Indeed, inhibition of Na+-K+-ATPase and, hence, a decrease in sodium tubule transport by the kidney is associated with increased rates of gluconeogenesis [9]. These findings suggest that energy-requiring processes, such as renal growth, sodium transport and gluconeogenesis compete for the available energy in nephron tubules and could explain the striking natriuresis observed under chronic acidosis [1]. Since respiration is the main source of energy supply for renal ion tubule transport, the altered renal Na+ and K+ handling under acidotic conditions may result from alterations in mitochondrial bioenergetics.

The present study examined energy-linked functions (respiration, electrochemical membrane potential and coupling between respiration and ADP phosphorylation) of kidney and liver mitochondria isolated from rats at 48 h after NH4Cl-induced metabolic acidosis.



   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 References
 
Experimental design
The experiments performed in male Wistar–Hannover rats (200–250 g) allowed free access to water and normal rat chow. The general guidelines established by the Brazilian College of Animal Experimentation (COBEA) were followed throughout the investigation. Metabolic acidosis was produced by substituting 0.25 M NH4Cl for drinking water. All animals drank ad libitum and the volumes ingested were recorded. Only those rats that drank nearly equivalent amounts of tap water or NH4Cl were used. NH4Cl-treated rats were maintained on their respective regimens for up to 2 days. The experiments were performed in parallel for each group of control and acidotic rats.

Kidney and liver mitochondria isolation
After 48 h of 0.25 M NH4Cl drinking, mitochondria were isolated from the whole kidneys (cortex and medulla) and liver of adult Wistar rats by conventional differential centrifugation [10]. Briefly, rat kidneys were rapidly removed, finely minced and homogenized in ice-cold buffer containing 250 mM sucrose, 0.5 mM EGTA and 10 mM HEPES buffer (pH 7.2). The mitochondrial suspension was then centrifuged at 500 g for 7 min, the resulting supernatant was centrifuged at 7800 g for 10 min and the pellet was re-suspended in the EGTA-free buffer at 6000 g for 10 min. Mitochondrial protein concentration was determined by the Biuret method [11], modified by the addition of cholate [12]. Rat kidney and liver (0.5 mg/ml) were added to the standard reaction medium containing 40 µM arsenazo III.

The experiments were performed at 28°C in standard medium containing 125 mM sucrose, 65 mM KCl, 1 mM MgCl2, 2 mM Pi, 10 mM HEPES, 5 mM succinate (pH 7.2) and 17 µM Ca2+ as determined by atomic absorption. Oxygen consumption experiments were performed in this same standard medium in the presence of 0.5 mM EGTA. All the assays performed using kidney mitochondria were compared with those using liver mitochondria in the same experimental groups.

Measurements of mitochondrial respiration
Oxygen consumption was measured in a 1.3 ml thermostated water-jacketed vessel equipped with a magnetic stirrer, using a Clark-type electrode (Yellow Spring Instruments Company) connected to a recorder [13]. Rat Kidney mitochondria (RKM, 0.5 mg/ml) were added to the standard reaction medium set at 28°C. Respiration rates are given in natom O2 min–1 mg protein–1. Phosphorylating (state III) respiration was initiated by addition of 200 nmol ADP/mg protein. Phosphorylation efficiency (ADP/O ratio) was calculated from the added amount of ADP and total amount of oxygen consumed during state III. To study the effect of metabolic acidosis on uncoupling protein (UCP) activity, mitochondria respiration was tested in the presence of CAT (carboxiatractilosyde), GDP (guanosin di-phosphate) and BSA (bovine serum albumin).

Estimation of mitochondrial transmembrane electrical potential
Mitochondrial electrical transmembrane potential ({triangleup}{Psi}) was estimated by the safranine O method [14]. The binding of safranine to the polarized inner membrane is followed by changes in fluorescence emission at 586 nm. The extent of fluorescence decreases after FCCP (carbonylcyanide-4-trifluoromethoxy-phenylhydrazone) addition, which totally eliminates the H+ electrochemical potential and this, corresponds to {triangleup}{Psi}. The remaining safranine fluorescence is not related to {triangleup}{Psi}. Changes in safranine fluorescence were recorded using a model F-4500 Hitachi spectrofluorometer (Hitachi Ltd. Tokyo, Japan) operating at excitation and emission wavelengths of 495 and 586 nm, respectively, with slit widths of 5 nm.

Measurement of mitochondrial Ca2+ transport
Variations in free medium Ca2+ concentrations were examined by measuring changes in the absorbance spectrum of Arsenazo III, using an SLM Aminco DW 2000 spectrophotometer (SLM Instruments, Inc., Urbana, Ill, USA) set at the 675–685 nm wavelength pair [15].

Chemicals
Most of the chemicals, including ADP, catalase, cyclosporin A (CsA), dithiothreitol, EGTA, FCCP, HEPES, safranine O, succinate, ruthenium red, sucrose, KCl, MgCl2, phosphate, calcium and NH4Cl, were obtained from Sigma Chemical Company (St. Louis, Missouri, USA). All other reagents were commercial products of the highest purity grade available.

Statistical analysis
For comparison, results from each rat were taken from at least three independent experiments, performed in triplicate, using mitochondria isolated from that animal. All data are reported as means ± SD and analysed using appropriate ANOVA or Mann–Whitney tests. Post-hoc comparisons between selected means were performed with Bonferroni's contrast tests when initial ANOVA indicated statistical differences between experimental groups. Comparisons involving only two means within or between groups were made using a Student's ± test. A P value <0.05 was considered to indicate significance (INSTAT Software Inc. Richmond, CA, USA).



   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 References
 
Figure 1 shows succinate-supported respiration and the efficiency of oxidative phosphorylation of kidney (panel A) and liver (panel B) mitochondria isolated from control (line Con) and NH4Cl-treated (line Ac) rats. The resting respiration (state IV) was significantly higher in kidney mitochondria isolated from acidotic rats than controls. Accordingly, both respiratory control (in natom oxygen/mg protein/min) and ADP/O ratio were significantly lower in these mitochondria (RC = 2.6 ± 0.21, P = 0.0001; ADP/O = 1.11 ± 0.06, P = 0.01; n = 5) than in control mitochondria (RC = 3.31 ± 0.10, ADP/O = 1.40 ± 0.05, n = 5). In contrast, there were no differences in respiratory control or ADP/O ratio in liver mitochondria isolated from controls and acidotic rats (Figure 1, panel B).The differences in kidney mitochondrial coupling were further analysed by monitoring {Delta}{Psi} changes during ADP phosphorylation supported by succinate oxidation. Figure 2A shows that {Delta}{Psi} of acidotic kidney mitochondria was slightly smaller than that of control mitochondria and that the transient {Delta}{Psi} decrease associated with ADP phosphorylation was longer (160 ± 13 s) in kidney mitochondria isolated from NH4Cl-treated rats than in control (91 ± 8 s) mitochondria (n = 15, P = 0.0113), which is in agreement with the respiration experiments. In liver mitochondria, the changes in {Delta}{Psi} induced by ADP were identical in control and acidotic rats (Figure 2B) (n = 5, P = 0.100).


Figure 1
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  		Fig. 1. Effect of NH4Cl-induced metabolic acidosis on respiratory control of isolated rat kidney (panel A) and liver mitochondria (panel B). Mitochondria (0.5 mg/ml) were added to a standard reaction medium as described in ‘Materials and methods’. The arrows indicate the addition of 200 nmols ADP.

 

Figure 2
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  		Fig. 2. Effect of metabolic acidosis on membrane potential ({triangleup}{Psi}) of isolated rat kidney (panel A) and liver mitochondria (panel B). Mitochondria (0.5 mg/ml) were added to a standard reaction medium as described in ‘Material and methods’ containing 5 µM Safranine O. The arrows indicate the additions of 200 nmols ADP and 0.5 µM FCCP.

 
In order to ascertain a possible involvement of UCP in the altered respiration of mitochondria from the acidotic rats, we measured respiration in media containing carboxyatractyloside (CAT, inhibitor of the ADP/ATP carrier), GDP (inhibitor of UCP) and BSA (chelator of fatty acids). Table 1 shows that the resting respiration (state IV) of mitochondria isolated from the acidotic rats (NH4Cl) was faster in the presence of CAT but decreased progressively after the sequential additions of GDP and BSA, thereby attaining a value equivalent to the control respiration. These results suggest that the lower efficiency of oxidative phosphorylation in the acidotic rats can be explained at least in part by a higher activity of the UCP, although the quantities of UCP in both sets of mitochondria were below the threshold of western blot using anti-UCP2 antibody and colorimetric analysis. This is in agreement with previous reports [16].


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  		Table 1. Effect of the UCP inhibitors GDP and BSA on respiration of mitochondria isolated from acidotic (NH4Cl) and control (Control) rats

 
Changes in Ca2+ handling by kidney mitochondria isolated from rats treated with NH4Cl
It is known that NH4Cl treatment in whole animals or in intact cells leads to a rise in intracellular free calcium concentration [Ca2+] [17]. The molecular mechanisms underlying the alterations in Ca2+ homoeostasis, induced by NH4Cl, seem to vary according to tissue, but its occurrence in cells suspended in Ca2+-free medium suggests the participation of Ca2+ release from intracellular pools [18]. Therefore, we compared Ca2+ handling by kidney and liver mitochondria isolated from controls and NH4Cl-treated rats. Figure 3 shows that the rate of Ca2+ uptake by mitochondria isolated from NH4Cl-treated rats was faster than in control rats and that mitochondria from the acidotic rats had the ability to buffer external free Ca2+ at concentrations significantly lower ({Delta}Ca2+ = 0.50 µM) than in control mitochondria (n = 5, P = 0.001). In order to understand the mechanisms underlying the differences in Ca2+ set points established by these mitochondria, we made further additions of Ca2+ or EGTA after the Ca2+ transport had attained a steady state. We found that Ca2+ uptake or release followed these additions, respectively, tending to restore the original steady-state level. These experiments suggest that both sets of mitochondria are able to establish stable Ca2+ set points and have the ability to accumulate and retain additional quantities of the cation (10 nmol Ca2+ mg/protein). In these experiments, the initial free Ca2+ concentration in the medium was 17 µM, as determined by atomic absorption and the calibration was performed by the additions of known amounts of EGTA [19]. In contrast, the experiments depicted in Figure 4 show that Ca2+ handling by liver mitochondria was similar in acidotic and control rats.


Figure 3
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  		Fig. 3. Succinate-supported Ca2+ uptake by kidney mitochondria from control and acidotic rats. The ordinates represent arsenazo III differential absorption spectrum (675–685 nm) and show the changes in free Ca2+ concentrations caused by the addition of 15, 20 and 25 µM EGTA. The free Ca2+ concentrations were 2.40 µM, 0.45 µM and 0.19 µM, respectively, as calculated by Inesi et al. [19]. Rat kidney mitochondria (RKM) (0.5 mg/ml) were added to a standard reaction medium as described in ‘Material and methods’ that contained 40 µM arsenazo III. The arrows indicate additions of 5 µM of CaCl2 and 5 µM EGTA.

 

Figure 4
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  		Fig. 4. Succinate-supported Ca2+ uptake by rat liver mitochondria (RLM) from control and acidotic rats. The ordinates represent arsenazo III differential absorption (675–685 nm). RLM (0.5 mg/ml) were added to the standard reaction medium containing 40 µM arsenazo III. The arrows indicate the addition of 0.5 µM of FCCP.

 
Figure 5 shows that similar results were obtained when these mitochondria were challenged by higher Ca2+ concentrations from the beginning of the experiments. In this setting, the addition of ruthenium red (RR), which inhibits Ca2+ uptake through the uniporter, under steady-state conditions, provided evidence that the rates of net Ca2+ release were similar in both mitochondria and were not significantly stimulated by NaCl, which may promote Ca2+ release via a mitochondrial Na+/Ca2+ exchanger. The subsequent addition of FCCP promoted a fast release of the remaining mitochondrial Ca2+ via reversal of the uniporter due to {Delta}{Psi} elimination. In contrast, the experiments aimed at measuring the initial rates of Ca2+ influx, after linearization of the Ca2+ tracings, confirmed that Ca2+ accumulation by mitochondria isolated from the acidotic rats was faster than in controls (265.1 ± 71.5 vs 152.9 ± 40.5 nmol Ca2+/mg mitochondrial protein/min, n = 5, P = 0.016).


Figure 5
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  		Fig. 5. Effects of ruthenium red, NaCl and FCCP on Ca2+ release from control and acidotic rat kidney mitochondria (RKM). RKM (0.5 mg/ml) were added to the standard reaction medium containing 24.5 µM CaCl2. The arrows indicate additions of 0.5 µM ruthenium red (RR), 10 mM NaCl and 0.5 µM FCCP.

 
To further show that this difference in Ca2+ set points was not the consequence of lower capacity of the control mitochondria to retain Ca2+ due to opening of the low conductance state of the permeability transition pore [20], we carried out experiments in the presence of the mitochondrial permeability transition pore inhibitor cyclosporin A [21]. Under these conditions, we again found that kidney mitochondria isolated from acidotic rats took up Ca2+ faster and established a set-point at a lower concentration of free Ca2+ than control mitochondria (Figure 6).


Figure 6
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  		Fig. 6. Effect of cyclosporin A on Ca2+ transport rat by kidney mitochondria (RKM) from control and acidotic rats. RKM (0.5 mg/ml) were added to the standard reaction medium containing 27 µM CaCl2 and 1 µM CsA. The arrows indicate additions of 0.5 µM FCCP.

 


   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
References
 
Studies in animals have shown that metabolic acidosis causes several biochemical changes in the kidney that are ultimately associated with organ hypertrophy [22–24]. In this regard, we have previously shown that chronic metabolic acidosis, caused by NH4Cl feeding, leads to nephron hypertrophy and to a decreased water-salt reabsorption by the kidneys [1,25,26]. Since renal ion tubule transport is highly dependent on mitochondrial energy and because mitochondria have been implicated in a variety of metabolic disorders, we examined mitochondrial energy-linked functions in chronic metabolic acidotic rats. The present results demonstrated that whole kidney mitochondria isolated from these rats showed higher resting respiration and, consequently, lower respiratory control and ADP/O ratio when compared to control mitochondria. These alterations were attenuated by the additions of GDP and BSA, suggesting the involvement of uncoupling proteins in the lower efficiency of oxidative phosphorylation in mitochondria isolated from the acidotic rats (Table 1). This was additionally supported by the slightly lower {triangleup}{Psi} and longer transient decrease in {triangleup}{Psi} that occurred during ADP phosphorylation (Figure 2A). No significant differences in either {triangleup}{Psi} or respiration were detected between control and acidotic liver mitochondria, attesting the organ specificity of these acidosis effects (Figure 2B).

Other organ-specific effects of NH4Cl-induced chronic acidosis included alterations in mitochondrial Ca2+ transport, such as increased rates of initial Ca2+ uptake and lower extra mitochondrial Ca2+ concentration under mitochondrial steady state conditions (Ca2+ set point) (Figure 5). The Ca2+ set point is established by a steady-state condition in which the rates of Ca2+ influx through the uniporter is equal to the rate of Ca2+ release through the Ca2+/H+ plus the Ca2+/Na+ antiporters. Therefore, an increased rate of Ca2+ influx, without a corresponding increase in the rate of Ca2+ efflux, explains the observed shift in Ca2+ set point in the acidotic rat mitochondria. This possibility is also supported by the lack of CsA effect on Ca2+ handling in both control and acidotic mitochondria. These findings also rule out the participation of mitochondrial permeability transition to explain the differences in Ca2+ transport between control and acidotic kidney mitochondria (Figure 6).

In NH4Cl-treated rats, it has been shown that an increased Ca2+ influx through the plasma membrane, in response to intracellular alkalosis, explains the higher cytosolic Ca2+ concentration [27]. This occurs in rat parotid [28], aortic endothelium [29], canine renal [30], bovine anterior pituitary [31] and rat lachrymal [32] cells. It is also known that, in addition to increases in cytosolic Ca2+, ammonia induces an increase in oxygen radical (ROS) production [33]. Since the combination of Ca2+ and ROS can elicit mitochondrial dysfunction, which is mediated by membrane thiol group protein oxidation (permeability transition pore formation) and lipid peroxidation [34], the present results may represent a mitochondrial response to counter Ca2+ and ROS build-up [35]. This is because the changes in Ca2+ handling may represent an adaptation of mitochondria towards the maintenance of intracellular Ca2+ homoeostasis by removing excess Ca2+ from the cytosol. Such an action would increase the intramitochondrial Ca2+ concentration that promotes stimulation of ROS generation at the level of the respiratory chain [36]. Secondly, increased ROS generation stimulates UCP activity, which stimulates respiration. This increase in respiration decreases the concentration of reduced respiratory chain components that may act as an electron donor to molecular oxygen to thereby control the rate of ROS generation [37]. ROS are ultimately derived from molecular oxygen, either as a byproduct of mitochondria respiration where O2– generation increases with PO2, as the product of specific oxidases such as NADPH, NADH oxidase or xanthine oxidase, or by interaction with various cellular constituents such as Fe2+, whereby OH– is generated from H2O2 in the Fenton reaction. Therefore, the availability of O2 may regulate ROS production according to the functional Km of the specific system involved. Recent studies have examined in vivo PO2 in the kidney and its effects on ROS generation to address the hypothesis that PO2 may limit oxidative stress in the kidney. Renal oxygen usage (QO2) normally increases linearly with tubule sodium reabsorption (TNa) above a basal level [38]. The slope of this line (TNa: QO2), above basal level, defines the efficiency with which the kidney uses O2 for chemical work. This efficiency depends markedly on ROS generation [39]. However, Laycock and colleagues [39] reported that the kidney increases QO2, while it reduces the glomerular filtration rate associated with decreases in TNa, producing a sharp reduction in TNa: QO2. Thus, enhanced mitochondrial O2 usage associated with increased urinary sodium excretion, as a consequence of oxidative stress, could account for the inefficient utilization of O2 by the acidotic kidneys in the present study.

In response to an acid insult, the kidney undergoes several adaptive changes in its structure, metabolism and function. It also appropriately increases acid excretion in the urine. The majority of these adaptive changes occur in the proximal tubule, which exhibits an increase in H/HCO3 exchange, ammoniagenesis and HCO3– generation, as well as a reduction in the reabsorption of inorganic phosphate and sulfate, which together contribute to increased acid secretion in the collecting duct. Metabolic acidosis is also, associated with the down-regulation of organic anion pathways, Na-dependent inorganic sulfate and Na-phosphate cotransporters. Interestingly, despite these adaptive and appropriate changes, the proximal tubule exhibits a significant reduction in salt and water transport in response to metabolic acidosis [2,25,26,40]. A defect in salt and water reabsorption in the proximal tubule produces an increase in fluid delivery and increased hydroelectrolyte reabsorption in Henle's loop, the distal tubule and the collecting duct system. A recent study demonstrated that metabolic acidosis exerts dual effects on urinary Na+ excretion [40]. The first is an early (~24 h) natriuresis resulting from decreased Na+ reabsorption in the proximal tubule and an Sgk1-related decreased epithelial sodium channel (ENaC) in the distal and collecting tubule [40]. Sgk1 is also known as an aldosterone-induced kinase and plays an important role in the regulation of both the expression and the activity of ENaC. The second effect, after 5 days of acidosis, is an aldosterone-induced upregulation of both Sgk1 and EnaC, which probably contributes to the antinatriuretic phase of metabolic acidosis [40]. Since we performed the present experiments using whole kidney structure, our studies were not specifically designed to determine the effects of metabolic acidosis on Na+ transport mechanisms along particular nephron tubule sites nor to correlate Na+ patterns with altered solute and water tubule handling. Nevertheless, previous experiments demonstrating altered renal sodium handling [25,26] may provide evidence for a reciprocal relationship between the level of efficiency of renal mitochondrial activity and ion tubule transport. Therefore, we hypothesize that the mild uncoupling and higher capacity for Ca2+ accumulation represents an adaptation of the mitochondria to cope with conditions of oxidative stress and high cytosolic Ca2+, which are associated with NH4Cl toxicity in renal cells. In addition, the decreased efficiency of oxidative phosphorylation may explain, at least in part, the striking natriuresis observed under chronic acidosis.



   Acknowledgments
 
Grants from CNPq (No. 500868/91-3), PRONEX (0134/97) and FAPESP (00/12216-8) supported this work. The authors wish to thank Ms Elizabeth C. Cambiucci for expert technical assistance.

Conflict of interest statement. None declared.



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 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received for publication: 23.11.06
Accepted in revised form: 23. 4.07


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