Abnormal erythrocyte choline transport in patients with chronic renal failure

Transcription

1 Clinical Science (1991) 80, Abnormal erythrocyte choline transport in with chronic renal failure F. C. FERVENZA', D. MEREDITH2, J. C. ELLORY2 AND B. M. HENDRY3 'Renal Unit, Churchill Hospital, Oxford, 'University Laboratory of Physiology, Oxford and 3Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, U.K. (Received 1 March/20 July 1990; accepted 10 September 1990) SUMMARY 1. Erythrocyte choline transport has been studied in nine on maintenance haemodialysis for chronic renal failure, six on continuous ambulatory peritoneal dialysis, 31 with renal transplants and in nine normal control subjects. 2. The mean maximum rate of choline influx (V,,,,,, measured at an extracellular choline concentration of 250 pmol/l) was 66.7 (SD 14.1) pmol h-' 1-' cells in on haemodialysis, 87.8 (SD 18.5) pmol h-' I-' cells in on continuous ambulatory peritoneal dialysis and 30.5 (SD 4.9) pmol h-' 1-' cells in control subjects. The increase in choline flux in on haemodialysis and on continuous ambulatory peritoneal dialysis compared with control subjects was highly significant (P< 0.001). 3. Renal transplant showed variable values for the Vmm, of choline influx (range pmol h-' 1-' cells). The values showed a sigmfcant negative correlation with creatinine clearance and this correlation correctly extrapolated to the maximum choline flux in normal subjects and in on dialysis. 4. The kinetics of choline transport have been studied in erythrocytes of on haemodialysis and control subjects in 'zero-trans' conditions after depletion of intracellular choline. The mean Vmm, in these conditions was 38.4 (SD 4.6) pmol h-' 1-' cells in on haemodialysis compared with 14.2 (SD 3.7) pmol h-' 1-' cells in control subjects. The mean K, under 'zero-trans' conditions yas 19.4 (SD 2.4) pmol/l in on haemodialysis and 7.4 (SD 1.4) pmol/l in control subjects. These differences were significant (P < 0.001). Key words: choline, dialysis, erythrocyte, membrane transport, uraemia. Correspondence: Dr B. M. Hendry, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K. Abbreviations: ACEI, angiotensin-converting enzyme inhibitor; CAPD, continuous ambulatory peritoneal dialysis; HD, haemodialysis; Mops, 4-morpholinepropanesulphonic acid; ZT, 'zero-trans'. INTRODUCTION Abnormal erythrocyte membrane transport of ions and amino acids has been reported in renal failure [l-31. The extent to which these alterations in erythrocyte membrane transport are representative of other cell types is controversial, but some evidence points to parallel changes in other tissues [4-61. The pathophysiological significance of these alterations in membrane transport remains unclear, and it is not established whether cell membranes are affected by circulating plasma factors in renal failure [l, 21. The membrane transport of choline is altered in lithium therapy and in Alzheimer's disease [7, 81. This paper reports a study of the membrane transport of choline in erythrocytes from with renal failure. The erythrocyte membrane possesses a high-affinity selective choline-transport system which has been wellcharacterized from a kinetic standpoint [9, lo]. The transport of choline into cells is vital for the synthesis of phospholipids and for the production of acetylcholine in excitable tissues. The neuronal uptake of choline also involves a specific membrane transport system; this neuronal transport is distinct from the erythrocyte choline transporter in that it is partially sodium-dependent. Nevertheless, the erythrocyte choline transporter is useful as a starting point for the study of choline transport in renal failure. The erythrocyte choline-transport system can operate in a choline-choline exchange mode or it can carry a unidirectional flux of choline 19, 101. Experimentally, the influx of choline into fresh human erythrocytes is partially via choline-choline exchange; this component of flux can be removed if erythrocytes are depleted of intracellular choline before the measurements ['zero-trans' (ZT) condi-

2 138 F. C. Fervenza et al. Table 1. Some clinical and laboratory parameters of the control subjects and patient groups studied Values are means with ranges in parentheses. Age (years) Plasma creatinine (rmol/l) Plasma urea (mmol/l) Plasma K+ (mmol/l) Haemoglobin (g/dl) Mean cell volume (fl) Control subjects HD CAPD Transplant (n=9) (n = 9) (n=6) (n=31) 43 (26-62) 104 (72-123) 4.1 ( ) 4.2 ( ) (43-68) 859 ( ) 29.4 ( ) 5.7 ( ) 8.0 ( ) 95.6 ( ) 55 (21-78) 97 1 ( ) 24.4 ( ) 5.2 ( ) 8.2 ( ) 91.6 ( ) 46 (20-67) 226 (88-846) 14.6 ( ) 4.4 ( ) 11.9 ( ) 94.3 ( ) tions]. In this work erythrocytes have been studied both immediately after initial washing and after establishing ZT conditions. The detailed kinetic analysis of choline transport was studied in ZT conditions as these results do not depend on the (variable) initial intracellular choline concentration. The ZT data can therefore be used to determine the apparent affinity constant of the choline receptor on the outside of the cell membrane. METHODS Patients Nine on maintenance haemodialysis (HD), six on continuous ambulatory peritoneal dialysis (CAPD) and 31 with renal transplants were studied and compared with nine normal control subjects. Patients with essential hypertension, diabetes mellitus or dementia and those on lithium therapy were excluded. Details of drug therapy in the patient groups were as follows. All HD were on vitamins B and C, folk acid and iron supplements. In addition, four were on B- blockers, two were on angiotensin-converting enzyme inhibitors (ACEI) and eight were on alfacalcidol. Of the six CAPD, two were on B-blockers and two were on ACEI. The medications of the transplant were: prednisolone (31), azathioprine (17), cyclosporin A (22), ACEI (1 l), B-blockers (13), diuretics (nine), cotrimoxazole (seven) and hydralazine (three). Some clinical and laboratory details of the subjects are shown in Table 1. Blood was taken from the HD immediately before a dialysis. The work has been approved by the Oxford Ethical Committee and each patient gave their consent. In the transplant creatinine clearance was measured using a single 24 h urine collection and a blood sample on the day of an outpatient visit. The same blood sample was used for the studies of choline transport. These measurements were made in transplant with stable plasma creatinine. Chemicals ['4C]Choline (specific activity 107 mci/mmol) was obtained from New England Nuclear (Stevenage, Hem, U.K.). Choline was purchased from Sigma Chemical Co. (bole, Dorset, UX.) and was recrystallized from ethanol. Other chemicals were Analar grade. Procedures A 5 ml sample of blood was taken from each subject into a heparinized tube and kept on ice for use within 2 h. Choline uptake was measured using established methods for erythrocyte transmembrane flux experiments [ 111. Erythrocytes were separated and washed by centrifugation (3000g, 5 min) in ice-cold saline of composition (mmol/l): NaCI, 140; KCI, 5; glucose, 5; 4-morpholinepropanesulphonic acid (Mops), 10; ph 7.4. Duplicate aliquots of cells were then resuspended in 1.0 ml of cold saline, at a packed cell volume of , containing 250 pmol/l choline and tracer amounts of ['4C]choline. The cells were incubated at 37 C for 5 min in this solution and incubation was stopped on ice. The cells were then rapidly separated from the supernatant by four centrifugation-washes in ice-cold magnesium chloride solution (107 mmol/l MgC1,-10 mmol/l Mops, ph 7.4). The cells were then lysed with 0.5 ml of 0.1% (v/v) Triton X-100 and the protein was precipitated with 1 ml of 5% (w/v) trichloroacetic acid. The 14C content of the clear lysate was measured by liquid scintillation counting in Pico- Fluor 40 scintillation fluid in a Packard Tri-Carb 2000 CA counter. Haemoglobin was determined spectrophotometrically in triplicate using Drabkins reagent, assuming the absorbance of packed cells to be 247 [111. Experiments were performed to establish that choline uptake was a linear function of incubation time up to 10 min. It was also established that cholie uptake was saturable as a function of extracellular choline concentration and that V,,, for uptake could be measured with an

3 Erythrocyte choline ti *ansport in renal failure 139 extracellular choline concentration of 250 pmolll. The data from the 5 min incubations were therefore used to calculate the maximum initial rates of choline uptake. A kinetic analysis of choline uptake was performed on erythrocytes from the HD and from the control subjects. After the initial wash, erythrocytes were resuspended in saline at a low packed cell volume (<0.025) and incubated at 37 C for 4 h to allow depletion of intracellular choline and create ZT conditions for measurement of choline influx. This procedure removes the trans-stimulation of choline uptake and therefore reduces V,,,. It was determined that a new steady-state V,,, was established after the 4 h depletion time. The cells were then washed again in ice-cold saline and resuspended in duplicate tubes in saline containing choline at pmol/l, in the presence of tracer amounts of [14C]choline. ChoLine influx was then determined with a 5 min incubation at 37 C as described above. These experiments allowed the calculation of the concentration-dependence of choline influx in ZT conditions. Statistical analysis Statistical differences between groups were analysed by analysis of variance and where appropriate further examination by application of Student's t-tests, but significance was not altered by using non-parametric tests. The Spearman test was used to assess the significance of correlations between measured parameters. RESULTS The rates of erythrocyte choline uptake from saline containing 250 pmol/l choline are shown for each subject studied in Fig. 1. These data are estimates of V,,, for the choline transporter in erythrocytes studied immediately after initial washing. The mean V,, in normal subjects was 30.5 (SD 4.9) pmol h-' 1-I cells compared with 66.7 ff * 3 I1 * : 0 1 Control HD CAPD RT Fig. 1. Individual values of V,,, for erythrocyte choline uptake from saline containing 250 pmol/l choline. Abbreviation: RT, renal transplant. Means k SD are also shown. Statistical significance: *P < compared with control subjects. The renal transplant group were also significantly different from control subjects (P < 0.05). i : (SD 14.1) pmol h-' I-' cells in HD and 87.8 (SD 18.5) pmol h-i I-' cells in CAPD. There was no overlap between the individual results in normal subjects (all <40 pmol h-' I-' cells) and in dialysis (all > 50 pmol h-' I-' cells) and the HD and CAPD groups were significantly different from the control subjects) (P < ). The values of V,,, in transplant were very variable (range pmol h-i I-' cells) and the individual values are shown in Fig. 1. There was no significant correlation between age and V,,, in any of the groups studied. The transplant studied had wide differences in renal function. Fig. 2 is a plot of V,,, against creatinine clearance in these. There was a significant negative correlation (P = 0.002, r = ) and the regression line is shown. There were also significant correlations between V,,, and serumcreatinine(p=o.oll, r=0.284) and serum urea (P=0.016, r=0.353). There was no significant correlation between V,,, and any of the other parameters in Table 1 (all P> 0.05). Erythrocytes from HD and control subjects were studied in ZT conditions and choline influx was measured as a function of extracellular choline concentration. The mean results for each group are illustrated in Fig. 3. The data can be fitted with Michaelis-Menten kinetics [ll] to provide an estimate of V,,. and of the apparent dissociation constant of the transporter (K,) in ZT conditions. The fits are shown by the lines in Fig. 3; the fitted value of V,=, in HD was 38.4 pmol h- ' I-' cells compared with 14.2 pmol h-' I-' cells in control subjects in ZT conditions. The value of K, was 19.4 pmol/l in HD and 7.4 pmol/l in control subjects under ZT conditions. The differences between control subjects and HD were significant (P< 0.001). DISCUSSION These results demonstrate clear abnormalities of erythrocyte cholie transport in on dialysis for chronic renal failure. These exhibit an increased capacity for choline uptake. The increase appears greater in on CAPD than in those on HD. Kinetic analysis of the choline transporter in haemodialysis under ZT conditions shows an increased V,,, and a reduced apparent affinity for choline binding compared with control subjects. Renal transplant exhibit variable erythrocyte choline uptake, but uptake does correlate inversely with creatinine clearance. This correlation (Fig. 2) can be extrapolated to creatinine clearances of 0 and 100 ml/min to produce V,,, values of 56.8 and 23.4 pmol h- ' 1-I cells, respectively. These values are in broad agreement with the data obtained for dialysis and control subjects (Fig. l), suggesting that a consistent relationship between renal function and choline transport exists in all the groups studied. Of the 31 transplant studied, 19 had been transplanted for over 6 months. Separate analysis of this group and of the remaining 12 showed no differences from the overall picture of Fig. 2. There were too few results from recently inserted functional transplants for

4 140 F. C. Fervenza et al. a LE Creatinine clearance (ml/min) Fig. 2. Correlation between V,,, for choline transport and creatinine clearance in renal transplant. The linear regression line (r= -0.55, P=0.002) is V,,, = (clearance). At a clearance of 100 ml/min, the line extrapolates to a V,,,, of 23.4 pmol. h-' I-' cells. conclusions to be drawn on the rate of recovery of choline transport abnormalities after successful transplantation. It is therefore not clear whether these alterations are a result of circulating factors acting directly on the mature erythrocytes, or whether they arise from factors affecting erythrocyte development in the bone marrow. Experiments on with acute renal failure and sequential studies on early successful transplantation could shed further light on this. The human erythrocyte choline transporter is known to cycle faster in the exchange mode than in ZT operation [9]. The observation that mean Vmu, results were reduced in ZT conditions in comparison with the values for immediate use of the same erythrocytes was therefore as expected. The increase in choline influx in the presence of intracellular choline is referred to as 'trans-stimulation'. Kinetic analysis of the choline transporter in ZT conditions confirmed the raised V,,, for choline transport found in cells from HD used immediately, and demonstrated abnormal choline binding affinity. This suggests that the abnormalities cannot simply be ascribed to an altered number of choline transport units per erythrocyte. There appear to be changes in the characteristics of the individual transporters. It is possible that recruitment of a new low-affinity transporter in addition to the existing high-affinity system could explain the results; alternatively, the existing transporter might be induced to alter in substrate affinity. Changes in erythrocyte amino acid transport by systems y+, ASC and gly, including altered substrate affinity, have been reported in HD [3, 12, 131. These amino acid transporters are primarily responsible for the transport of lysine and arginine (y+), alanine, cysteine and serine (ASC), and glycine (gly). Such widespread alterations in membrane transport could arise through alterations in the membrane lipid environment of the transporters: certain phospholipid species and free fatty acids are altered in uraemia both in plasma and in erythrocytes. It has been suggested that these substances Extracellular choline concn. (pmol/l) Fig. 3. The mean initial rates of erythrocyte choline influx under ZT conditions as a function of extracellular choline concentration in HD (m) and control subjects (0). The error bars are SEM. The lines are Michaelis- Menten fits to the data with the values of V,,, and K, under ZT conditions reported in the text. alter membrane Na+,K+-adenosine triphosphatase pump function [14, 151 and they could equally well affect other membrane transport systems. A striking feature of the results for choline transport is the lack of overlap in the Vmu, for dialysis and for control subjects (Fig. 1). This clear separation has not been found in studies of any other transport system in renal failure. The correlation of transport function with irenal function in the transplant population has also not been demonstrated for other transporters. This clear distinction between erythrocytes from normal subjects and uraemic should expedite studies of the origins of the altered transport of choline. The clinical importance of altered membrane choline transport is unclear. The neuronal uptake of choline is partially Na+ -dependent, unlike the erythrocyte transporter, and the two choline transport systems are clearly not identical [9]. The erythrocyte defect in Na+,K +-adenosine triphosphatase pump activity in renal failure appears to be representative of other tissues, but other reported erythrocyte membrane transport alterations have not been described elsewhere [ 1, Neuronal choline transport could be unaffected in renal failure. If neurons and other cells do exhibit abnormal uptake of choline then there is the possibility that this might alter cell function both through altered acetylcholine levels and via altered phosphatidylcholine and sphingomyelin synthesis. There are reports of altered choline transport in erythrocytes and cultured neurons during lithium therapy and in Alzheimer's disease [7, 81, but the pathophysiological significance of these changes is also unknown. ACKNOWLEDGMENTS This work was supported by the National Kidney Research Fund. F.C.F. is supported by CNPq (Brazil). We thank Dr D. 0. Oliver and Professor P. J. Morris for permission to study under their care.

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