Intraplatelet calcium levels in patients with acute renal failure before and after the administration of loop diuretics

Ilona R. Shilliday1, and Marjorie E. M. Allison1,2

1 Renal Unit, Glasgow Royal Infirmary 2 Department of Medicine, University of Glasgow, Scotland, UK



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Intracellular calcium [Ca]i has been found to be elevated in hypoxic cells in vitro and in erythrocytes and lymphocytes from patients who are septic. Loop diuretics decrease [Ca]i in platelets from patients with hypertension and in red blood cells from normal volunteers. We report the results of a study designed to measure [Ca]i in platelets from patients with acute renal failure (ARF) before and after the administration of loop diuretics.

Methods. Sixteen healthy adults and seven patients with ARF were enrolled into the study. Intraplatelet calcium was measured using a fluorescent probe (quin2). Patients with ARF all received intravenous (i.v.) dopamine, 2 µg/kg body weight, and 20% mannitol, 100 ml every 6 h and, in double-blind manner, either torasemide, frusemide, or placebo, 3 mg/kg body weight i.v. every 6 h. Data from subjects given either frusemide or torasemide have been considered together and termed the diuretic group.

Results. Basal levels of [Ca]i in platelets from patients with ARF were significantly higher than in controls (126.9±35.7 nmol/l vs 85.7±22.2 nmol/l, P=0.02), but were not affected by the administration of loop diuretic (126.9±35.7 nmol/l vs 165.9±49.7 nmol/l, P=0.09, pre- vs post-diuretic).

Conclusions. Intraplatelet calcium is raised in patients with ARF. Loop diuretics have no significant effect on intraplatelet calcium in these patients.

Keywords: acute renal failure; arginine vasopressin; dopamine; intraplatelet calcium; loop diuretics; mannitol; quin2



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Elevated free cytosolic calcium ([Ca]i), found in cells injured by ischaemia has been proposed as one of the mechanisms important in the pathogenesis of ischaemic cell injury [14]. High [Ca]i causes an uncoupling of mitochondrial oxidative phosphorylation, resulting in reduced ATP stores [5]. Wilson et al. [5] showed that accumulation of intra-mitochondrial calcium occurs following reperfusion of ischaemic renal tissue and that there is a highly significant correlation between raised mitochondrial calcium concentration and decreased mitochondrial respiration. High [Ca]i also causes activation of phospholipases, resulting in an alteration in membrane permeability and membrane enzyme activity [68]. Activation of intracellular proteases in ARF in the presence of raised [Ca]i will convert xanthine dehydrogenase to xanthine oxidase, which in turn leads to the production of hydroxyl radicals and tissue injury [9]. In addition calcium has been shown to potentiate the oxygen free radical-mediated injury to mitochondria [10].

Two clinical studies have shown that sepsis increases [Ca]i in erythrocytes and lymphocytes [11,12]. Loop diuretics have been shown to cause a significant decrease in [Ca]i in platelets from patients with essential hypertension and in red blood cells from normal volunteers [13,14]. In an experimental model of ischaemic ARF, loop diuretics prevented the rise in mitochondrial calcium normally seen in oliguric ARF [15].

We therefore measured free cytosolic calcium levels in platelets from patients with ARF, before and after the administration of a loop diuretic. Our aim was to answer the following questions:

Is intraplatelet calcium raised in patients with ARF compared to healthy control subjects?
Do loop diuretics have any effect on intraplatelet calcium levels in patients with ARF?



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Before starting the study, informed written consent was obtained from the normal volunteers and from the patients or, if consciousness was impaired, from their next of kin.

Normal volunteers
Sixteen healthy adult volunteers were entered into the study (10 males and six females). All subjects had normal renal function, had no history of acute or chronic illness, and none was taking any medication. The mean age of controls was 30 years (range 25–40 years).

On the first study day (control) at 1300 hours a 16-G indwelling plastic cannula was inserted into the forearm, the tourniquet removed, and 45 ml blood taken into sodium citrate for measurement of [Ca]i in platelets. The following day (day 2) at 1000 hours an intravenous injection of frusemide 20 mg/2 ml, or torasemide 10 mg/2 ml, or placebo 2 ml was given. The study was double blind and neither the investigator nor the subject was aware of what was being given. Three hours later a further 45 ml blood was taken into sodium citrate as before for measurement of [Ca]i. During the second study day patients were encouraged to drink freely to prevent dehydration. Estimation of [Ca]i took place immediately after venepuncture.

A 1-week wash out period was allowed and days 1 and 2 repeated 1 week and 2 weeks later. Each subject therefore received each of the loop diuretics and placebo in a random order.

Acute renal failure patients
Seven patients with ARF participated in this study: two females and five males; mean age 60 years (range 46–69 years). All patients were part of a larger, double-blind study assessing the effect of loop diuretics on the clinical outcome of ARF. Forty-five millilitres of blood was taken for measurement of baseline intraplatelet calcium, as for the normal volunteers. Patients were then randomly allocated to receive frusemide, torasemide, or placebo 3 mg/kg over 1 h every 6 h (given in a double-blind manner) together with a continuous i.v. infusion of low-dose dopamine and 20 g mannitol i.v. over 1 h every 6 h. Five of the patients received a loop diuretic, two received placebo. Twenty-four hours later a further 45 ml of blood was taken for estimation of intraplatelet calcium.

Measurement of intraplatelet calcium
[Ca]i was measured using a fluorescent probe: quin2 acetoxymethylester [16]. Platelet-rich plasma (PRP) was prepared by collecting whole blood into 3.9% sodium citrate (9 ml whole blood/1 ml sodium citrate), and centrifuging at 250 g for 5 min at room temperature. PRP was incubated at 37°C for 30 min with quin2 (2 µl quin2 were added to each millilitre of PRP). Ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA; 5 mmol/l; Fluka) was added to bind to any extracellular calcium, and PRP then centrifuged at 250 g for 10 min at room temperature. The supernatant was removed leaving a pellet of platelets containing quin2. The pellet was resuspended in platelet buffer titrated to pH 7.3 at 37°C. The buffer was calcium free to prevent platelet aggregation during centrifugation. The platelets in the suspension were counted in a Coulter counter (Technicon H-1TM system). The concentration of platelets was adjusted to 1.5–2x108/ml and 2 ml of the suspension was dispensed into each of nine cuvettes and placed in a water bath at 37°C.

The first cuvette was placed into the spectrofluorometer and the background fluorescence was measured with a Perkin–Elmer LS-3B spectrofluorometer at 339 nm excitation and 492 nm emission; 20 µl of 0.1 mol/l CaCl2 was then added to the cuvette. In platelets from healthy controls, there should be no change in the fluorescence signal, since the added calcium should not leak across the plasma membrane into the platelets (Figure 1aGo).



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Fig. 1. (a) Fluorescence signal from platelets from a healthy volunteer. (b) Fluorescence signal from platelets from a patient with ARF.

 
The platelets were disrupted with 20 µl of digitonin, thereby releasing the intracellular quin2, which then binds to the added calcium, giving rise to the maximum fluorescence signal, Fmax. The quin2 is then fully saturated; 20 µl of MnCl2 was added; manganese displaces calcium from the quin2 and therefore gives rise to a very low signal, Fmin.

The intraplatelet calcium concentration can then be calculated using the equation:


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where 115 is the equilibrium dissociation constant in nmol/l, F the fluorescence of the intact cell platelet suspension, Fmax the maximum fluorescence after the platelets have been disrupted with digitonin (20 µl), and Fmin the minimum fluorescence obtained after addition of 20 µl 2 mmol/l MnCl2.

Effect of arginine vasopressin
Arginine vasopressin (AVP) stimulates influx of calcium from the suspension into the platelets as well as a release of intracellular stores of calcium into the cytosol. As quin2 is only found inside the platelets, the fluorescence signal obtained after addition of AVP gives an indication of total intracellular calcium.

In cuvette 2 the response of platelet [Ca]i to 20 µl AVP (1 µmol/l) was measured in the presence of 1 mmol/l calcium (20 µl of 0.1 mol/l CaCl2 added to the suspension). The fluorescence signal obtained was therefore due to influx of calcium from the suspension and release of intracellular stores. This new level of fluorescence from intact calls was used to calculate [Ca]i.

Effect of AVP and EGTA
The extracellular calcium concentration was then reduced to negligible concentrations in cuvette 3 by adding 50 µl of 200 mmol/l EGTA. In the presence of EGTA, there is no calcium influx from the suspension into the cell and the fluorescence signal obtained when AVP is added comes solely from release of intracellular stores into the cytosol. Once again the new F was used to calculate [Ca]i.

Each stage was repeated twice in the remaining six cuvettes and [Ca]i is expressed as the mean of triplicate measurements.

Statistical evaluation
Results are expressed as mean±SD. Differences in means were calculated by Student's t-test.



   Results
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 Subjects and methods
 Results
 Discussion
 References
 
Table 1Go shows the baseline values for intraplateletcalcium in normal volunteers and in patients with ARF.


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Table 1. Intracellular calcium in platelets from ARF patients vs controls (before loop diuretic)

 
Basal levels of [Ca]i were significantly higher in ARF subjects than in normal volunteers.

There was no significant change in [Ca]i after the addition of AVP, or after the addition of AVP/EGTA in either group.

In the five ARF patients who received loop diuretic there were no significant changes in basal levels of [Ca]i nor in the response to AVP (Table 2Go).


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Table 2. Intracellular calcium in platelets from ARF patients, (A) before and (B) after a loop diuretic

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This is the first study to report the measurement of intracellular calcium in platelets from patients with ARF. Basal levels of [Ca]i are significantly higher than in normal volunteers and there is evidence to suggest that in these patients the intracellular stores of calcium are depleted.

The absence of a rise in [Ca]i after AVP may be because quin2 in the cytosol was already bound to calcium that had (a) leaked in from outside the cell, and (b) leaked from intracellular stores. The lack of rise in [Ca]i after EGTA/AVP would imply that the stores of calcium were already depleted. Depletion of intracellular stores of calcium may reflect alteration of membrane phospholipids in very sick patients. The platelet cell membrane was clearly abnormally permeable to calcium as the intracellular fluorescence signal rose after the addition of extracellular calcium (Figure 1bGo). It is known that ischaemia alters the composition of plasma membrane phospholipid [6,7]. This would allow influx of extracellular calcium into the cytosol as well as efflux of intracellular stores of calcium. A reduction in Ca ATPase activity due to reduced ATP levels would reduce calcium efflux from the cell [18].

Todd and co-workers demonstrated a significant rise in free cytosolic calcium in erythrocytes following endotoxin (40.451±2.923 nmol/l (before) vs 84.523± 5.019 nmol/l (after), P<0.001) [12]. A similar rise occurred in septic patients (septic 96.261±7.511 nmol/l vs non-septic 45.376±2.925 nmol/l, P<0.001) [12]. However, others did not find an increase in free intracellular calcium levels in lymphocytes following endotoxin and tumour necrosis factor (TNF), although free intracellular calcium levels in lymphocytes were significantly higher (P<0.05) in septic patients (176±12 nmol/l) compared to non-septic head-injured (110±11 nmol/l), cardiac surgical (112±9 nmol/l), or healthy controls (112±5 nmol/l) [11].

Platelets from normal volunteers behaved as expected, with a rise in [Ca]i after AVP due to an influx of calcium across the plasma membrane and release of intracellular stores of calcium into the cytosol (Figure 1aGo). In the presence of EGTA, and therefore the absence of extracellular calcium, the increase in the fluorescence signal was less, coming solely from released intraplatelet stores of calcium.

The administration of loop diuretics have been shown to reduce levels of calcium in platelets from hypertensive patients [13]. We could not detect this response to loop diuretics in patients with ARF. It is possible that the alteration in membrane structure due to phospholipase activity affects the binding sites of drugs, resulting in reduced drug action. Also, the effect of administering loop diuretics prior to the onset of the insult resulting in ARF is unknown.

Volunteers in the control group were significantly younger than patients in the two study populations. However, basal levels of [Ca]i in normal volunteers in this study are not different from those found in normal subjects in a previous study looking at platelet cytosolic free calcium in essential hypertension where the age range of subject was 31–68 years [13]. Age itself probably has no effect on intraplatelet levels of calcium, although hypertension does. None of the normal volunteers had hypertension.

Direct measurement of [Ca]i in renal tubules in a clinical setting is not possible and a suitable alternative is needed. White blood cells, red blood cells, and platelets are easily accessible. We elected not to use white blood cells, as the cell population is not homogenous. Haemoglobin in red blood cells interferes with the fluorescence of quin2. Platelets have some features in common with vascular smooth-muscle cells, namely a calcium-dependent contraction-coupling mechanism and {alpha}2-adrenoceptor-operated calcium channels [19,20]. As with vascular smooth muscle, angiotensin II and vasopressin may also promote calcium influx [21]. Thus, platelets are a good model to study in clinical ARF to assess the possible role of intracellular calcium in the initiation (vascular) phase of ARF. Similar changes occurring in tubule epithelial cells may contribute to cell death and the maintenance of ARF.



   Notes
 
Correspondence and offprint requests to: Dr I. R. Shilliday, Renal Unit, Western Infirmary, Glasgow G11 6NT, UK. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 27. 9.99
Revision received 10.10.00.