Vol. 40, No. 6, December 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Poges

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1 Vol. 40, No. 6, December 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Poges MODULATION OF OUABAIN SENSITIVE SODIUM POTASSIUM PUMP OF ERYTHROCYTES FROM PATIENTS WITH CHRONIC RENAL FAILURE : ROLE OF ACUTE HEMODIALYSlS R. PRASAD*, R. MOND, S. JAIN 1, G. KAUR AND K.S. CHUGH 2 Departments of Biochemistry, Internal Medicine I and Nephrology 2, Postgraduate Institute of Medical Education and Research, Chandigarh , (INDIA) Received September 25, 1996 SUMMARY Significantly higher levels of plasma urea creatinine and potassium were observed in patients with renal failure compared to normal controls. The RBC sodium concentration was raised whereas the RBC potassium concentration was decreased in chronic renal failure. These alterations in the RBC Na f af~d K J concentrations were associated with decrease in ouabain sensitive sodium efflux rate and ouabain sensitive sodium efflux rate constant. However, there was no significant impact of acute hemodialysis on the intracellular electrolytes levels, ouabain sensitive sodium efflux rate and ouabain sensitive sodium efflux rate constant. These findings suggest an intrinsic alteration in the transport capacity of Na +-K + pump which could account for the rise in intracellular sodium and fall in intracellular potassium content in the RBCs of chronic renal failure patients. INTRODUCTION Challenges to sodium and potassium homeostasis are manifold and require mechanisms to cope with both surfeit and deficit of total body sodium and potassium. The concentration of sodium inside the cell and the transport of Na = and K + across the cell membranes are largely mediated by a specific transport system termed the Na+-K + pump (1-4). Several authors have reported an alteration in the RBC sodium content in patients with chronic renal failure and have provided evidence for a defect in the sodium transport of erythrocytes (5-7). *Corresponding Author /96/ /0 Capyright 1996 by Academic Pres.~ Au.~tralia. All rights af reproduction in any forra reserved.

2 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Earlier studies to delineate the mechanisms of increase in intracellular sodium have been limited to the measurement of pump mediated sodium efflux, the passive sodium leak and Na+-K + ATPase activity (8-10). The exact mechanism responsible for the alteration in sodium and potassium homeostasis in patients with chronic renal failure remain unclear. To detect the alterations, we studied the concentration of red cell sodium as a measure of ouabain sensitive efflux (EOS). EOS when expressed as a fraction of the original red cell sodium concentration, gives a measure of the ouabain sensitive sodium efflux rate constant (ERCSos), were measured by a non-radioactive technique as described by Cumberbatch and Morgan (11). This technique measures only the net ouabain-sensitive sodium transport. In addition the impact of hemodialysis was assessed on the sodium efflux rate and efflux rate constant in the RBC of patients with chronic renal failure. MATERIAL AND METHODS Cations contents and sodium transport were studied in the uremic RBCs obtained from the 15 male patients with chronic renal failure. The etiology of chronic renal failure was chronic glomerulonephritis; benign nephrosclerosi.~, diabetic nephropathy and chronic interstitial nephritis. The ellect ol ucute hemodialysis on cation content and sodium transport in RBCs of patients with chronic renal failure was also assessed. The age range of the patients included in this study was 32 to 58 years (mean ± SD ). all control subjects were normotensive with no significant medical problem. Fresh venous blood was drawn from the patients and controls in heparinized tubes, centrifuged at 3000 g. Plasma and buffy coat were removed by aspiration. RBCs were washed four times with a washing solution containing 152 mm choline chloride and 1 mm MgCI 2 buffered with 10 mm tris Hepes (ph 7.4) at 4 C in cold centrifuge. Measurement of intracellular and plasma cations Washed RBCs were completely lysed in double distilled water. After centrifugation, the sodium and potassium concentrations in the supernetant were measured by flame photometry. Plasma sodium and potassium levels were also measured by the same technique. 1088

3 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Measurement of plasma urea and creatinine Plasma urea and creatinine were measured on autoanalyser (Technicon- SAM-II). Measurement of ouabain sensitive efflux rate and efflux rate constants of sodium in RBCs The ouabain sensitive sodium efflux rate and efflux rate constant in tile RBCs were measured using a new, simple and non-radioactive technique described by Cumberbatch and Morgan (11). 20 pl of ouabain 10.2 mol I 1 in 80% ethanol was added to each aliquot contains 3.5 ml of heparinized venous blood from patients of chronic renal failure and the healthy controls to give a final concentration of approximately 10.4 tool I "1. The samples were shaken at 37 C for one hour. Two samples to which only 20pl of 80% ethanol had been added, were shaken at 37 C served as controls, the ouabain sensitive effluxes were than calculated from the increase in Na]RB C between O and 1 hr of incubation. Efflux rate [ESos ] [Na]RBC = = mmol/i cells/h Incubation time (h) Efflux rate constant (ERCSos ] ESos [Na]RBC = h-1 [Na]WRBc = Sodium content of washed rbc at zero hour Statistical Analysis Results were expressed as means+s.d. Statistical significance was determined using Student's unpaired t-test between the groups and Student's paired t-test within the groups. RESULTS Urea, creatinine and potassium were significantly higher in chronic renal failure patients in comparison to healthy control subjects (Table 1). The mean sodium concentration expressed as millimoles per litre of cell was significantly higher as compared to the mean value of healthy control (10_ 1.0 retool/i). Mean potassium content in the RbCs of chronic renal failure patients was significantly lower as compared to the controls (Table 2). 1089

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL TABLE 1 : Plasma biochemical parameters in chronic renal failure and normal healthy subjects Parameter _.C hron_ i.c_re.ha_l_ fa_ i_lu r_e g_rou_p_... Control group Before hemodialysis After hemodialysis Urea 162.0± 17.O 100.0:1: (mg/dl) Creatinine 10.0 ± ± (mg/dl) Sodium ± (meq/i) Potassium ± (meq/i) The values are expressed as mean :I:S.D. in 15 subjects of the chronic renal failure and 10 normal controls p < 0.05 was considered as significant Ouabain sensitive sodium efflux rate and efflux rate constants are summarized in Table 2. There was significant reduction in ouabain sensitive sodium efflux rate in the RBCs of patients with chronic renal failure compared to the controls. A significant reduction was also observed in ouabain sensitive sodium efflux rate constant in the RBCs of chronic renal failure patients compared to the controls. The reduction in ouabain sensitive sodium efflux rate constant in RBCs of chronic renal failure patients was in proportion to the reduction of ouabain sensitive efflux rate in these patients. Whereas hemodialysis with low potassium solution in chronic renal failure patients fully corrected the hyperkalemia, there was no significant effect on intracellular RBCs sodium and potassium contents before and after acute hemodialysis. Further, we did not find any significant change in ouabain sensitive sodium efflux rate and rate constants in RBCs of chronic renal failure patients before and after dialysis. 1090

5 BIOOHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL DISCUSSION In the present study, an alteration was observed in the distribution of electrolytes (Na +, K +) between the plasma and intracellular fluid of RBCs in patients with chronic renal failure. This imbalance of electrolytes is primarily attributed to the defect in ouabain sensitive Na + K + pump which controls the homeostasis of sodium and potassium intracellularly. Increased level of sodium and decreased levels of potassium in the RBCs of chronic renal failure patients are associated with decrease in ouabain sensitive sodium efflux rate. It has been suggested that certain factors in the uremic plasma may lead to a decrease in the activity of Na-K pump. In an earlier study, [Na+-K +] ATPase activity of normal erythrocytes exposed to uremic plasma was 16% lower than that of normal erythrocytes exposed together. Krammer et al observed that Na efflux of normal erythrocytes was depressed by exposure to uremic plasma (9). it was TABLE 2: sodium, potassium, ouabain sensitive sodium efflux rate and ouabain sensitive sodium efflux rate constant in RBCs of chronic renal failure and control subjects Parameter _Chron ic_renaj_ f. a iju r_e _group... Before hemodialysis After Hemodialysis Control group RBC sodium 13.5_ (mmol/i cells) RBC potassium O+4.0 (mmol/i cells) Efflux rate 2.0_ _+O _+0.2 [ESSos ] nmol/i cells/h Efflux rate O. 17 _ constant ERCSos (h"1) The values are expressed as mean + SD in 15 subjects of the chronic renal failure and 10 normal controls P<0.05 was considered as significant. 1091

6 Vol. 40, NO. 6, 1996 BIOOHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL therefore suggested that accumulation of uremic toxins inhibits the cellular Na +- K 4 ATPase, resulting in abnormalities in the cellular cation content (12). A digoxin-like substance has been postulated to accumulate in the plasnla of uremic patients (1 3). Such a substance could bind tightly or irreversibly to the membrane of uremic erythrocytes and modulate the Na-K pump activity. However, the exact nature of the toxin present in uremic plasma has not yet been characterized. No significant change was observed either in the sodium and potassium levels or ouabain sensitive sodium efflux rate in the RBCs of chronic renal failure patients after acute hemodialysis. Although, hyperkalemia was fully corrected in patients on hemodialysis, the latter was not always associated with improvement in the Na-K pump activity. The persistence of the defect in Na-K pump on dialysis may reflect inadequate removal of putative uremic toxin responsible for this abnormality. This possibility is supported by the partial correction of plasma urea and creatinine on dialysis in uremic patients. On the other hand, reduction in the potassium content in the leukocytes (12) and muscle cells (14) in uremic patients can be normalized by hemodialysis. Serum potassium concentration in chronic renal failure is maintained within normal limits both by renal and extra renal adaptive mechanisms ( ). Despite a marked decrease in glomerular filtration rate, renal adaptation provides for a rapid increase in potassium excretion per surviving nephron to the extent that potassium excretion may exceed filtered load in patients with a major decrease in functional renal mass (17-19). However, the capacity of the surviving nephrons to excrete an additional acute potassium load is impaired (1 8). Therefore, the observed hyperkaiemia in chronic renal failure is suggestive to the presence of an increase excretory burden, possibly due to ingestion of potassium rich foods and medications, since it is corrected by hemodialysis with low potassium solutions. 1092

7 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL In addition to the decrease in ouabain sensitive sodium efflux rate in tile RBCs of patients of chronic renal failure, a decrease n ouabain se~sitive sodiuni efflux rate constant was found in the RBCs of patients of chronic renal failure. It can be inferred that low turnover rate of Na-K pump which may lead to diminished sodium efflux and increase in the intracellular Na + concentration and decrease in the potassium concentration in the RBCs in chronic renal failure. The turnover of Na+-K + pump may also be influenced by intracellular sodium itself (20,21). Alternatively the increase intracellular RBC sodium could result from increase in sodium influx in chronic renal failure, but earlier studies have shown that there is no difference in the sodium influx in the uremic and normal erythrocytes (9). Acknowledgements This work was sponsored by Potash and Phosphate Institute of Canada and India Programme, Sector 19, Delhi-Gurgaon Road, Gurgaon. We also thank Dr. G. Dev, Director, Potash and Phosphate Institute of Canada for his invaluable interest and encouragement during the entire period of this study. REFERENCES 1. Glynn, I.M. (1962). Brit. Med. Bull. 24, Reddy, P.C. (1992). J. Neurol Transm. (Gen. sect.) 89, Cumberbatch, M. and Morgan, B. (1981). Clin. Sci. 60, Whittman, R. and wheeler, K.D. (1970). Annu. Rev. Physiol. 32, Cheng, J.T., Kahn, T. and Kaji, D.M. (1984). J. Clin. Invest. 745, Swaminathan, R., Clegg, G., Cumberbatch, M., Zarelan, Z. algol Mackenna, F. (1982). Clin. Sci. 62, Corry, D.B., Tuck, M.L., Brickman, A.S., Yanagawa, N. and Lee, D.B.N. (1986). Kid. Int. 29, Cole, C.H., Bafe, J.W. and Welt, L.G. (1968). Trans. Assoc. Am. Physicians 81,

8 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 9. Kramer, H.J., Gosopodinov, D. and Kruch, F. (1976). Nephron. 16, Edmonson, P.S., Hilton, P.J., Jones, N.F., Patrick, J. and Thomas, R.D. (1975). Clin. sci. Mol. Med. 49, Cumberbatch, M. and Morgan, B. (1978). Clin. Chim. acta 89, Patrick, J. and Jones, N.F. (1974). Clin. Sci. Mol. Med. 46, Graves, S.W., Brown, B. and Valdes, R. Jr. (1983). Ann. Intern. med. 99, Cole, C.H. (1973). Clin. Sci. Mol. Med. 45, Bank, N. and Aynedjian, H.S. (1973). J. Clin. Invest. 52, Mitch, W.E. and Wilcox, C.S. (1982). Am. J. Med. 72, Schultze, R.G. (1971). J. Clin. Invest. 50, Keith, N. (1943). Arch. Intern. reed. 71, Leaf, A. and Camara, A.A. (1949). J. Clin. Invest. 28, Sach J.R. (1970). J. Gen. Physiol. 56, Knight, A. and Welt, L.G. (1974). J. gen. Physiol. 63,