L-type calcium current of isolated rat cardiac myocytes in experimental uraemia

Paul Donohoe, Aisling C. McMahon1, Omal V. Walgama2, Federica Bertaso, Mark E. C. Dockrell, Hilary A. Cramp1, Adrian M. Mullen1, Michael J. Shattock3, Bruce M. Hendry and Andrew F. James

Department of Renal Medicine, GKT School of Medicine, King's College London, 1 Anthony Raine Research Laboratories, St. Bartholomew's Hospital, Queen Mary Westfield College, 2 Cardiac Surgical Research and 3 Cardiovascular Research, The Rayne Institute, Centre for Cardiovascular Biology and Medicine, St. Thomas' Hospital, King's College London, London, UK



   Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. End-stage renal failure is associated with a low-output cardiomyopathy, left ventricular hypertrophy and increased QTc dispersion. Cardiac dysfunction is prevalent in patients at the beginning of dialysis and is an important predictor of mortality. Ca2+ influx through voltage-gated L-type Ca2+ channels plays a key role in the excitation–contraction coupling of cardiac myocytes. The purpose of this study was to examine the effect of subtotal nephrectomy (SNx) in the rat on both cardiac L-type Ca2+ currents and action potential duration.

Methods. Wistar rats underwent two-stage SNx; control rats (C) underwent bilateral renal decapsulation. Animals were sacrificed after 8 weeks, and ventricular myocytes were isolated. SNx rats showed a 2-fold increase in plasma urea and creatinine compared with C rats. Whole-cell patch clamp techniques were used to examine L-type Ca2+ channel currents in isolated cardiac myocytes at 37°C. In separate experiments, the epicardial monophasic action potentials of isolated perfused whole hearts from C and SNx rats were recorded.

Results. The amplitude and current–voltage relationships of the L-type Ca2+ current were not significantly different in myocytes from C (n=11) and SNx (n=8) rats. However, the rate of inactivation of the Ca2+ current was increased by ~15–25% (P<0.05) in myocytes from SNx rats. The action potential duration (APD33) at the apex of the left ventricle was ~20% shorter (P<0.01) in hearts from SNx rats as compared with controls.

Conclusions. Renal failure is associated with rapid inactivation of cardiac ventricular myocyte L-type Ca2+ currents, which may reduce Ca2+ influx and contribute to shortening of the action potential duration.

Keywords: cardiac action potential duration; isolated cardiac myocytes; L-type Ca2+ currents; partial nephrectomy model; uraemic cardiomyopathy; whole-cell patch-clamp



   Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
End-stage renal failure is associated with a 10-fold increase in age-matched cardiovascular mortality [1] and a cardiomyopathy associated with low output left ventricular failure [2], left ventricular hypertrophy [3], myocardial fibrosis [4] as well as increased intra-ECG variation in QT interval (‘QT dispersion’), which has been associated with an increased risk of sudden death [5]. The aetiology of this cardiomyopathy is complex, but is likely to include hypertension, anaemia, secondary hyperparathyroidism, acidosis and uraemic toxins. The cardiomyopathy is prevalent in patients just beginning regular dialysis therapy and is an important indicator of subsequent mortality [6].

It is clear from animal work that renal failure is associated with cardiac contractile dysfunction, even at the level of the single cell [7,8]. Previous work using subtotal nephrectomy (SNx) models of chronic uraemia in the rat has demonstrated a 25% reduction in cardiac output of isolated perfused hearts from SNx rats compared with hearts from sham-operated controls [7]. More recently, isolated ventricular myocytes from the same animal model have been studied. A reduction in both the amplitude and velocity of contraction of isolated cardiac myocytes from SNx rats has been demonstrated [8]. Both the reduction in cardiac output and the reduction in contractile amplitude of isolated cardiac myocytes following SNx were associated with a reduced positive inotropic response to external Ca2+, suggesting a possible change in Ca2+ handling by cardiac myocytes following SNx [7,8]. We previously have examined systolic and diastolic Ca2+ levels in electrically paced isolated cardiac myocytes in SNx and control rats. While diastolic Ca2+ levels appeared to be elevated in myocytes from SNx rats, there was no change in the maximum systolic Ca2+ level [9–11]. Thus, SNx appears to be associated with abnormalities of Ca2+ handling and excitation– contraction coupling in the rat subtotal nephrectomy model.

Here we present data using whole-cell patch-clamp techniques to examine the L-type Ca2+ currents of isolated cardiac ventricular myocytes in the subtotal nephrectomy model of chronic renal failure in the rat. L-type Ca2+ channels are responsible for the vast majority of Ca2+ influx into cardiac myocytes following depolarization, and may be one of the components of excitation–contraction coupling affected by SNx. We also examined the effect of SNx on epicardial monophasic action potential duration in isolated perfused hearts.



   Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Surgery
Six-week-old male Wistar rats were anaesthetized and underwent SNx as a two-stage procedure as previously described [8]. Sham-operated control rats (C) underwent two-stage bilateral renal decapsulation. Following surgery, animals were pair-fed standard rat chow and housed in individual cages, with free access to water. Systolic blood pressure was measured by tail-cuff sphygmomanometry at 8 weeks after the second surgical procedure. The day following systolic blood pressure measurement, animals were sacrificed for myocyte isolation. At this time, blood samples were taken from the thoracic cavity and subsequently analysed for urea, creatinine and haematocrit. No animals were excluded on the basis of these blood samples.

Myocyte isolation
Ventricular myocytes were isolated by retrograde perfusion of the heart via the aorta with an enzyme-containing solution (collagenase class 2, Worthington, USA and hyaluronidase I-C, Sigma, UK), as previously described [8]. Cells were stored in 100 µM Ca2+-containing solution at room temperature, and used in patch-clamp experiments within 8 h of isolation.

Electrophysiological experiments
Isolated cells were perfused with Tyrode solution (in mM: NaCl 142.0, KCl 5.4, NaH2PO4 0.4, MgCl2 1.1, HEPES 10.0, glucose 5.5, taurine 10.0, CaCl2 1.0, pH 7.4 with NaOH) at 37°C and a flow rate of 2–3 ml/min. Myocytes suitable for patch-clamp experiments were Ca2+-tolerant, single, rod-shaped cells that ranged from 20 to 50% of isolated cells. Pipettes had a tip resistance of 2–4 M{Omega} when filled with the Cs+-rich pipette solution (in mM: L-aspartic acid 85, CsOH 85, TEA-Cl 20, HEPES 10, EGTA 1, MgCl2 2, MgATP 4, Na2GTP 0.1, Na2 creatine phosphate 5, pH adjusted to 7.4 with TEA-OH). In some experiments, the EGTA concentration of the pipette solution was 10 mM, as indicated in the text.

Whole-cell patch-clamp recordings were made using EPC-9 (HEKA GmbH, Germany) and Axopatch 200B (Axon Instruments Inc., USA) amplifiers. Data were acquired at 2 kHz to the hard disc of an Apple computer using Pulse/PulseFit software (HEKA GmbH, Germany). Series resistance and capacitance compensation were applied electronically. Voltage pulses (250 ms) between -60 and +60 mV were applied in 10 mV increments from a holding potential of -60 mV every 4 s (Figure 1AGo). Pre-pulses (30 ms) to -40 mV were used to inactivate voltage-dependent Na+ channels. Paired pulse protocols were applied to quantify the steady-state voltage dependence of Ca2+ channel inactivation. A series of 300 ms pre-pulses were applied every 4 s from -80 to +40 mV in 5 mV increments, followed by a 200 ms test pulse to -10 mV.



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Fig. 1. (A) Voltage-gated transient inward currents activated by voltages from -30 mV to positive. Voltage-clamp pulse protocol inset. (B) Complete block of inward current elicited following perfusion of 0.1 mM CdCl2. Vpulse=0 mV. (C) Current density–voltage relationships for calcium current in control myocytes (open symbols, n=11) and SNx myocytes (filled symbols, n=8). Peak inward current at start of pulse (circles), current at end of pulse (squares). Pipette [EGTA] was 1.0 mM.

 
Currents were normalized to cell capacitance (pA/pF). There was no significant difference in mean whole-cell capacitance between C (134±12 pF, n=11) and SNx (129±15 pF, n=8) myocytes. Leak current was subtracted from the current data, assuming an Ohmic leak resistance between -60 and -40 mV.

All data are expressed as mean±SE. Statistical significance was assessed by either two-tailed ANOVA or Student's t-test, and P<0.05 was considered significant. Curve fitting was performed using IgorPro Vers 2.1.

Epicardial monophasic action potentials
Control and SNx rats were prepared surgically as before. A total of five control and seven SNx rat hearts were used to record monophasic action potentials (MAPs), again at 8 weeks following the second surgical procedure.

Rats were anaesthetized with SalanaxTM (Pentobarbitone, Rhone Merieux) 0.1 ml/100 g intraperitoneally, and heparinized with 0.15 ml of 1 : 5000 heparin solution (Leo Laboratories, UK). Depth of anaesthesia was assessed by hind leg withdrawal reflex. Hearts were excised into ice-cold Krebs–Henseleit buffer (in mM: NaCl 119, KCl 4.7, MgSO4 1.2, H2PO4 1.2, NaHCO3 25, glucose 11.5, CaCl2 1.2 mM, pH 7.4 with 1 mM NaOH, bubbled with 95% O2–5% CO2 mix) and mounted on a Langendorff perfusion apparatus. Perfusion was commenced with Krebs–Henseleit buffer at 37°C in a retrograde manner with a perfusion pressure of 100 cmH2O, resulting in flow rates ranging from 10 to 15 ml/min. Hearts were unpaced and were allowed to beat spontaneously.

After a 20 min stabilization period, monophasic action potentials were recorded using an epicardial suction electrode, positioned in turn at the apex and base of the left ventricle, and at the right ventricle.



   Results
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The mean plasma urea (15.9±2.1 mM) and creatinine (108±11 µM) concentrations of the SNx rats in the present study corresponded approximately to a 2-fold increase compared with C rats (urea 6.5±0.2 mM; creatinine 65.6±3.1 µM), P<0.01 for both urea and creatinine. SNx rats were not significantly anaemic (haematocrit 0.29±0.02) compared with C rats (haematocrit 0.33±0.04). SNx rats did not show a significantly higher mean systolic blood pressure (94 mmHg) than C rats (100 mmHg).

Voltages from -30 mV to positive elicited rapidly activating and inactivating, time-dependent inward currents (Figure 1AGo) which were blocked by bath application of 0.1 mM CdCl2 (Figure 1BGo), consistent with the properties of cardiac L-type Ca2+ currents.

There was no significant change in the amplitude and current–voltage relationship of Ca2+ current following SNx (Figure 1CGo). The current–voltage relationship was U-shaped, and generally maximally inward at -10 mV for both control (-16.8±2.0 pA/pF) and SNx (-18.3±3.1 pA/pF) myocytes. There was also no difference in mean time-to-peak of Ca2+ current between SNx and C myocytes (4.6±0.5 and 4.9±0.5 ms at 0 mV respectively, Figure 2AGo).



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Fig. 2. (A) Mean time-to-peak for L-type Ca2+ current for control myocytes ({square}) and SNx myocytes ({blacksquare}). (B) Examples of normalized L-type Ca2+ currents recorded from both control and SNx myocytes, showing more rapid inactivation of currents from SNx myocytes. Vpulse=0 mV. Superimposed lines represent fits to a double exponential relation (see text). Pipette [EGTA] was 1.0 mM.

 
However, the inactivation of the Ca2+ currents was more rapid in SNx myocytes compared with C myocytes. After reaching a peak, the Ca2+ currents inactivated with a time course characteristic of cardiac L-type Ca2+ currents. The inactivation of the Ca2+ currents as a function of time (t) elicited by voltage pulses from -10 to +10 mV was fitted by a double exponential relationship, I(t)=A1 exp (-t/{tau}f )+A2 exp (-t/{tau}s), where {tau}f and {tau}s are the fast and slow time constants of inactivation, respectively (Figure 2BGo).

Neither {tau}f nor {tau}s showed significant voltage dependence between -10 and +10 mV (Figure 3AGo). SNx was associated with shortening of both {tau}f (C 17±0.8 ms; SNx 15±1.6 ms at 0 mV) and {tau}s (C 57±4.0 ms, SNx 46±2.0 ms at 0 mV) for all voltages from -10 to +10 mV (Figure 3AGo). The shortening of the slow time constant ({tau}s) was significant (P<0.05). SNx had no effect on the values of A1 or A2 (Figure 3BGo), and it was clear that the slow component of inactivation predominated over the fast component, as A2 was greater in magnitude than A1 for all voltages between -10 and +10 mV.



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Fig. 3. (A) Voltage dependence of {tau}s (circles) and {tau}f (squares) in control (open symbols) and SNx (filled symbols) myocytes. The asterisks indicate P<0.05 (ANOVA). (B) Voltage dependence of A1 (circles) and A2 (squares) in control (open symbols) and SNx (filled symbols) myocytes. Pipette [EGTA] was 1.0 mM.

 
Inactivation of the cardiac L-type Ca2+ current is known to be predominantly Ca2+ dependent. In view of this, the effect of increasing the intracellular Ca2+-buffering capacity was investigated in experiments using a pipette [EGTA] of 10 mM. Again, the Ca2+ currents inactivated with a double exponential time course. Figure 4AGo shows the values of {tau}f and {tau}s under these conditions. Broadly speaking, the range of mean control values of {tau}s (40–70 ms) was unchanged by increasing the pipette Ca2+-buffering capacity. However, in contrast to the results with 1 mM pipette [EGTA], {tau}s was voltage dependent between -10 and +10 mV (compare Figures 3AGo and 4AGo) with 10 mM EGTA in the pipette. Unexpectedly, the mean control {tau}f was faster when the pipette [EGTA] was increased. SNx was again associated with shortening of both {tau}f (C 12.5±1.3 ms, n=11; SNx 7.6±1.1 ms at 0 mV, n=8) and {tau}s (C 49.6±6.2 ms, SNx 41.6±3.0 ms at 0 mV) for all voltages from -10 to +10 mV. The shortening of {tau}f was significant (P<0.05, ANOVA between -10 and +10 mV).



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Fig. 4. (A) As in Figure 3AGo, but with a pipette EGTA concentration of 10 mM; n=11 C and 8 SNx. Asterisks indicate P<0.05. (B) Voltage dependence of Ca2+ current inactivation. Top, pulse protocol. Middle, example family of current traces (C myocyte). Bottom, relationship between pre-pulse voltage and normalized current during the second pulse; {circ} C (n=4), • SNx (n=6). Pipette [EGTA] was 10 mM.

 
The voltage dependence of Ca2+ current inactivation was examined using a two-pulse protocol (Figure 4BGo) and a pipette EGTA concentration of 10 mM. The relationship between pre-pulse voltage and peak Ca2+ current could be fitted by a Boltzmann equation: I/Imax=1/[1+ exp(( Vm-V0.5)/Vs]+c, where Vm is the membrane potential during the pre-pulse, V0.5 is the voltage of half-maximal inactivation and Vs is the slope factor. The fits to the inactivation of the Ca2+ current in SNx and C myocytes were virtually superimposed (Figure 4BGo), demonstrating that there was no significant alteration in the voltage dependence of inactivation following SNx. There was no significant difference in V0.5 between C (-25.9±0.5 mV, n=4) and SNx (-25.1±0.4 mV, n=6) myocytes.

The duration of the action potential in isolated perfused hearts from C (n=5) and SNx (n=7) animals was examined by monophasic action potential recordings from the epicardial surface of the ventricle. Figure 5Go shows example recordings of monophasic action potentials from the apex and the base of the left ventricle (LV) and from the right ventricle (RV). The mean action potential duration from each region of the heart, calculated as the time between the beginning of the upstroke and 33% repolarization of the peak of the action potential (APD33), are shown in Table 1Go. APD33 was shorter in the LV apex and RV as compared with the LV base. SNx shortened the APD33 by ~20% in the LV apex (P<0.05). There was no significant difference between SNx and control animals in the heart rate of isolated perfused hearts.



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Fig. 5. Monophasic action potentials recorded from the epicardial surface of isolated, perfused hearts from control and SNx rats. The action potential amplitude was measured as the difference between the diastolic voltage and the peak of the action potential. Dotted lines indicate the diastolic potential, the peak of the action potential and the voltage level at 33% repolarization of the action potential. The action potential duration (APD33) was measured as the time from the start of the upstroke (upward arrow) to 33% repolarization of the peak (downward arrow).

 

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Table 1. Heart rate and APD33 of isolated perfused hearts from control and SNx rats

 



   Discussion
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 Methods
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We have shown that the inactivation of the L-type Ca2+ current of isolated cardiac ventricular myocytes was more rapid in partially nephrectomized rats than in sham-operated controls, due to a shortening of slow and fast time constants of inactivation. SNx had no effect on the peak current–voltage relationship for L-type Ca2+ current. Renal failure is associated with myocardial hypertrophy, particularly of the left ventricle. We have shown previously by histological examination of sections of heart that relatively mild renal failure, as in the present study, is associated with left ventricular hypertrophy without significant increases in blood pressure [12]. The changes in L-type Ca2+ currents we have observed are quite different from those reported for other models of cardiac hypertrophy. Patch-clamp analysis of isolated ventricular myocytes from a rat model of renovascular hypertension demonstrated an increase in peak L-type Ca2+ current density, and slower inactivation [13]. An infarct-induced model of cardiac hypertrophy in the rat demonstrated a reduction in L-type Ca2+ current density, and slower inactivation [14]. A catecholamine-induced model of hypertrophy in the rat was associated with no changes in L-type Ca2+ current–voltage relationships, and no change in the inactivation time course [15]. Taken together, these facts suggest that the electrical manifestations of the cardiomyopathy associated with renal failure is distinct from hypertrophy associated with hypertension or infarction. The finding that the SNx rats were not hypertensive compared with C rats confirms that the described changes in L-type Ca2+ currents are associated with changes in renal failure other than hypertension. Although the SNx rats were mildly anaemic, our findings are not in keeping with the reduced L-type Ca2+ current density described in a rat model of iron deficiency anaemia [16]. The mean membrane capacitance of the SNx myocytes in the present study (129 pF) was not different from that of C myocytes (134 pF), suggesting that there was no difference in the membrane surface area between the SNx and C myocytes of this study. Measurements of cell length and diameter in a previous study suggest that cell volume was increased after SNx, consistent with myocardial hypertrophy associated with renal failure [17].

Cardiac L-type Ca2+ channels inactivate by Ca2+-and voltage-dependent mechanisms [18]. Ca2+-dependent inactivation ({tau} ~50 ms) is very much faster than voltage-dependent inactivation ({tau} ~500 ms), and predominates under physiological conditions when Ca2+ is the principle charge carrier [18]. Ca2+-dependent inactivation occurs as a result of Ca2+ binding to the {alpha}1c subunit of the channel in the micromolar range [19]. Calmodulin may contribute to the Ca2+-dependent inactivation of the L-type Ca2+ current [20]. Thus, the inactivation rate is dependent on the Ca2+ concentration in the immediate vicinity of the channel at the cytosolic surface of the membrane and is highly sensitive to Ca2+ influx through the channel and Ca2+ release from the sarcoplasmic reticulum [17]. Ca2+-dependent mechanisms most probably contributed to the L-type current inactivation reported here since Ca2+ ions were the principal charge carrier. However, increasing the intracellular Ca2+ buffering capacity by changing the pipette [EGTA] from 1 to 10 mM did not slow inactivation significantly and did not reveal the much slower voltage-dependent mechanism. Thus, even with 10 mM EGTA in the pipette, it was not possible to chelate cytosolic Ca2+ in the vicinity of the channel sufficiently to prevent Ca2+-dependent inactivation. This is consistent with the findings of other groups who also show Ca2+-dependent inactivation when 10 mM EGTA was used as the Ca2+ buffer in the pipette [21].

The mechanism of the effect of SNx on L-type Ca2+ current inactivation is unclear. Previous work has shown an increase in basal cytosolic Ca2+ concentration ([Ca2+]c) associated with SNx in isolated ventricular myocytes [9,10]. Since Ca2+-dependent inactivation is dependent on micromolar range changes in [Ca2+] in the locality of the channel, it is unlikely that these changes (C ~60 nM; SNx ~100 nM) in basal [Ca2+]c in the nanomolar range could have resulted in the speeding up of inactivation seen in our experiments. Since neither the peak Ca2+ current nor systolic Ca2+ release [10] are increased after SNx, it is unlikely that an increase in the [Ca2+]c at the cytosolic surface of the Ca2+ channel upon excitation contributes to the changes in inactivation in renal failure. Rather, it seems likely that a change in the Ca2+ sensitivity of the inactivation mechanism results in an increased rate of Ca2+ current inactivation after SNx.

The shortening of inactivation of the L-type Ca2+ current may be due to altered channel protein conformation or function as a result of changes in protein expression or post-translational modification. Recent evidence suggests the existence of alternative splice variants of the cardiac L-type Ca2+ channel {alpha}1c subunit and changes in the expression of splice variants in cardiomyopathy [21]. Changes in the expression of alternative splice variants of the third membrane-spanning domain of motif IV (or IVS3) of the {alpha}1c subunit have been shown in a model of myocardial infarction-induced hypertrophy in the rat [22]. Other splice variant isoforms of the {alpha}1c protein differing in domains known to be important in voltage- (IS6) and Ca2+-dependent (C-terminal cytosolic tail) inactivation mechanisms have been reported [23,24]. Thus, one explanation for the increased rate of inactivation might be the expression of splice variants in SNx experiments, particularly variants differing in the C-terminus of the {alpha}1c subunit. Alternatively, changes in binding of calmodulin may contribute to the changes following SNx [20].

Rapid inactivation of the Ca2+ current in uraemia can be expected to have two consequences for cardiac function: firstly, a reduction in the total amount of Ca2+ influx during depolarization. The observed shortening of the mean time constants of inactivation reduced the estimated total Ca2+ influx by 22%, from 4.62x10-7 mol/cm2 to 3.59x10-7 mol/cm2. A reduction in the Ca2+ influx after SNx might be expected to result in a reduced Ca2+ content of the sarcoplasmic reticulum and, thereby, less Ca2+ release on stimulation. However, we have shown previously that the Ca2+ transient was not affected and the resting cytosolic Ca2+ concentration was increased after SNx [9–11], an effect that cannot be accounted for by reduced Ca2+ influx through L-type Ca2+ channels. Taken together, these facts suggest that changes in other Ca2+ handling pathways (e.g. Na+/Ca2+ exchange, sarcoplasmic reticular Ca2+ sequestration, Ca2+ buffering) may be contributing to the abnormalities in Ca2+ handling in uraemia.

The epicardial action potential duration at 33% repolarization from the apex of the left ventricles was found to be significantly shortened by ~20% in hearts from SNx rats compared with C rats (Table 1Go). Rapid inactivation of the Ca2+ current may contribute to this action potential shortening after SNx. The action potential duration determines the refractory period and is important in maintaining normal rhythmic cardiac function. We have found recently that changes in the properties of a 4-aminopyridine-sensitive repolarizing K+ current may also contribute to changes in the action potential duration in renal failure [25]. Accordingly, the changes in ion currents, Ca2+ handling and excitation–contraction coupling of isolated cardiac myocytes in renal failure are the subject of further investigation.



   Acknowledgments
 
The authors would like to thank Yasunobu Okada (Okazaki, Japan) and Minoru Horie (Kyoto, Japan) for their comments on the manuscript. Paul Donohoe is supported by clinical PhD studentship (FS/96080) from The British Heart Foundation.



   Notes
 
Correspondence and offprint requests to: Dr A. F. James, Department of Renal Medicine, GKT School of Medicine, King's College London, Bessemer Road, London SE5 9PJ, UK. Back



   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
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Received for publication: 14. 4.99
Revision received 4. 2.00.