Energetics and specificity of transcellular NaCl transport in the dog kidney

Ini. J t3b~hcrr1.. Vol. 12. pp. 245 !o 250 0 Pergamon PM\ Lid 19X0. Prmkd m Great

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ENERGETICS AND SPECIFICITY OF TRANSCELLULAR NaCl TRANSPORT IN THE DOG KIDNEY FREDRIKKIIL, OLE M. SEJERSTED and PETTER A. STEEN University

of Oslo, Institute

for Experimental

Medical

Research,

Ullevaal

Hospital,

Oslo

I, Norway

Abstract-l. Ouabain inhibits NaCl reabsorption and oxygen consumption in the dog kidney as expected if transport of sodium by Na-K-ATPase across the peritubular cell membrane was the only energy-requiring transport. 2. The facilitated transport across the luminal cell membrane of NaCl is not specific for chloride, since ouabain and ethacrynic acid inhibit bromide and thiocyanate reabsorption as much as chloride reabsorption.

INTRODUCTION Na-K-ATPase bound to the cell membrane, pumps sodium out of cells, and in vivo and in vitro studies on many tissues indicate that three sodium ions are extruded for each ATP split (Bonting & Caravaggio, 1963; Sen & Post, 1964). The basolateral cell membrane of both proximal and distal tubules has a high content of Na-K-ATPase. The simplest conceivable model for transtubular reabsorption is a facilitated, non-energy requiring transport of sodium across the luminal cell membrane, which probably lacks Na-KATPase, and an energy requiring transport by Na-KATPase across the basolateral membranes. In this model sodium reabsorption varies with changes in the sodium concentration in the cytoplasm close to the basolateral membrane, On the assumption that 6 ATP are generated for each 02 consumed, the ratios between changes in sodium reabsorption and oxygen consumption would be ANa/A02 = 18. However, the energetic efficiency of tubular reabsorption of sodium in the kidney as a whole is higher, even if it is assumed that the energy requirements of other transport or metabolic processes do not rise when the tubular reabsorption is raised (Kjekshus et al., 1969). Studies on the whole kidney indicate that with regard to sodium reabsorption two major tubular cell populations with different characteristics exist. In one system, mainly localized in the proximal tubules, hydrogen ions are exchanged with sodium ions across the luminal cell membrane and the transcellular transport by Na-K-ATPase is restricted to NaHCO,. There is no transcellular NaCl transport in this system. However NaHCOB extruded into the intracellular space exerts an osmotic force which reabsorbs waterand Nail through the tight junction. This bicarbonate dependent reabsorption of water and NaCl in the proximal tubules does not require energy and accounts for the high ANa/A02 ratio for the whole kidney (Kiil er al., 1979). In the diluting segment, osmotic reabsorption can be excluded because the segment is watertight. In this 245

segment NaCl is reabsorbed by the transcellular route. This segment does not reabsorb NaHCO, whereas NaCl reabsorption can be stimulated by raising sodium concentration in the tubular fluid. Ouabain, a specific inhibitor of Na-K-ATPase, inhibits transcellular sodium reabsorption. In doses compatible with life, ouabain inhibits less than 40% of the total tubular sodium reabsorption during sodium loading. Sejersted et al. (1977) showed in experiments performed during mannitol diuresis in dogs, that NaCl reabsorption and oxygen consumption were reduced by ouabain corresponding to a ANa/AO, ratio of 14.5 f 1.3. Because of its vasoconstrictive effect, ouabain reduces glomerular filtration rate (GFR), and hence tubular reabsorption. Measurements were obtained at identical GFR by manipulating the renal perfusion pressure, but an unproven assumption is that the proximal bicarbonate dependent reabsorption of water and NaCl are equal before and after ouabain administration. These technical shortcomings can now be avoided. After administration of acetazolamide, bicarbonate dependent water and NaCl reabsorption stay constant over a wide range of GFR (Mathisen et al., 1979a). Thus, a reduction in GFR would have no effect on bicarbonate dependent reabsorption. Reductions in sodium reabsorption and oxygen consumption can be completely attributed to the inhibition of Na-K-ATPase activity. The results of such examinations on anaesthetized dogs are reported in the first part of the present study. A second important question concerning transcellular NaCl reabsorption is, whether the facilitated transport across the luminal cell membrane is specific for chloride (Kiil, 1978). To examine this question we have compared the inhibitory effect of ouabain and ethacrynic acid on chloride, bromide, thiocyanate and iodide reabsorption. Ethacrynic acid was chosen because it may specifically inhibit NaCl transport across the luminal cell membrane (Sejersted et al., 1978).

246

FREDRIK KIIL. OLE M. SUERSTED and PETTER A. STEEN MATERIALS

AND

METHODS

Experiments were carried out on mongrel dogs of either sex, weighing from 15 to 23 kg. They were exsanguinated for 300 ml, 2 days before the experiment. The plasma was separated from the red cells and reinfused to increase the renal arteriovenous difference in oxygen saturation (Kiil et al., 1961). Anaesthesia was induced with sodium pentobarbital 25 rng. kg-’ intravenously and maintained with additional doses as indicated. Muscle relaxation was obtained with pancuronium bromide 0.2 mg. kg- ’ doses of and maintained with additional 0.05 mg.kg- ‘. The dogs were intubated and ventilated with a volume ventilator (modified Cyclator Mark II) to keep a constant PcoZ close to 5 kP during the experiment. Polyethylene catheters were inserted in a femoral vein and artery for infusions, blood sampling and pressure recordings. The tip of the arterial catheter was located just below the origin of the renal arteries and the pressure was recorded by means of a Statham pressure transducer (P23Gb) and a Sanborn amplifier and recorder. The right kidney was exposed through a retroperitoneal flank incision. The renal vein was cannulated with a soft polyethylene catheter introduced through the contralateral femoral vein. A thin, polyvinyl catheter in the renal artery, the tip pointing upstream, served for infusion of acetylcholine and ouabain. An electromagnetic flowmeter probe (Nycotron, Oslo), fitting the renal artery snugly, permitted continuous recording of renal blood flow (RBF) on the Sanborn recorder. A nylon snare placed on the artery distal to the probe was used for complete occlusion of the artery 34 times during the experiment for zero adjustment of the flowmeter reading. The gain of the flowmeter was calibrated according to the RBF values obtained from haematocrit, para-aminohippurate (PAH) clearance and arteriovenous extractions of PAH during three control periods. The ureter was cannulated with a soft polyvinyl catheter. Body temperature was kept constant with the use of heating pads. A priming solution containing inulin (50 rng. kg b.w.- ‘), creatinine (0.04 rng. kg b.w.- i) and PAH (120mg) in 0.9% NaCl solution was injected intravenously, followed by sustained infusion at 2 ml.min-’ to obtain arterial plasma concentrations of 2&30 mg per lOOm1, 10-20 mg per 100 ml and l&2.5 mg per 100 ml respectively. NaBr** and Nal”’ were added to the injectate and infusate to give a stable plasma activity in the individual experiment with a range between experiments of 500& 20,000 counts/min. ml- l and 10,00@30,000 counts/ min. ml- ’ respectively. In the dogs given ethacrynic acid SCN 24 mg. kg-’ was added to the injectate and 0.14 rng. kg-’ .min- ’ added to the infusate. Before control clearance periods 1.5-2 I of 0.9% NaCl was infused intravenously in the course of one hour. The infusate was kept at body temperature, and after this initial volume loading it was infused slightly faster than urine losses. KCI 8-10mmol~l-1, CaCl* 3 mmol.l-‘, MgS04 2mmol.l-’ and KH2P04 1 mmol.l- ’ were added to all solutions, and additional KCI infused to keep plasma potassium concentration above 4 mmol . l- ‘. Plasma concentration of bicarbonate was kept constant by infusing NaHCO, if needed.

Acetazolamide (100 mg. kg b.w.- ‘) was infused intravenously in the course of 334 min. After three control clearance periods at steady urine flow, ouabain (90 pg. kg b.w.- ‘) was injected intrarenally in the course of a few minutes in six dogs. To counteract the renal vasoconstrictive effect of ouabain, acetylcholine was infused into the renal artery at a rate of 4 pg.rnin-’ starting before and continuing for at least 10 min after completion of ouabain infusion. These clearance periods were obtained when the haemodynamic changes induced by acetylcholine had subsided. In the middle of each clearance period before and after ouabain infusion, blood for oxygen determination was collected anaerobically from the artery and renal vein in silicone-treated glass syringes, and oxygen saturation was measured spectrophotometritally using microcuvettes (Refsum & Sveinsson, 1956). Haemoglobin concentration was measured in triplicate with the cyanmethemoglobin method (Hainline, 1958). Oxygen consumption was calculated as previously described (Kiil er al., 1961), but no corrections were made for the oxygen dissolved in urine and renal lymph. The six dogs not given ouabain received after control clearance periods, ethacrynic acid 3 mg. kg- ’ intravenously followed by a continuous infusion of 1.5 mg.kg-‘.hr-‘. Plasma and urine were analysed for inulin (Rolf et al., 1949) and PAH (Smith et al., 1945) Sodium and potassium were determined on a direct flame photometer using lithium as internal standard (Instrumentation Laboratories I.L. 343). Chloride was measured on a chloride titrator (Radiometer CTN 10). Br*’ and 1” activities were determined in a well-scintillation counter (Selektronik). Blood gases were determined with I.L. electrodes. SCN was determined colourimetrically by an automated modification of Pettigrew and Fell’s method (Pettigrew & Fell, 1972). RESULTS

Energy requirement for transcellular NaCl reabsorption Table 1 summarizes the effects of acetazolamide and ouabain on renal haemodynamics, electrolyte reabsorption and oxygen consumption. The changes in RBF and GFR induced by ouabain administration were not significant, whereas sodium and chloride reabsorption and renal oxygen consumption were approximately halved. Bicarbonate reabsorption was slightly but significantly reduced. On the assumption that both the inhibition of NaCl reabsorption and NaHC03 reabsorption were due to reductions in transcellular energy requiring reabsorption, the rates between reductions in sodium and oxygen consumption averaged ANa/AOl = 17.8 + 1.5. However, if ouabain inhibited the proximal tubular NaHC03 reabsorption and the bicarbonate dependent water and NaCl reabsorption, two molecules NaCl should be subtracted for each molecule NaHC03 inhibited (Mathisen et al., 1979b). If this assumption is correct, the average ANa/AOz = 16.6 f 1.5. None of these ratios are significantly different from 18. Specificity of transcellular NaCl reabsorption Table

2 summarizes

fractional

reabsorption

of

154 &9 19 * 10 NS

Ouabain (9Opg-kg b.w.-*) Difference P

294 + 80 12 +_57 NS

283 + 41

RBF (mf.min-‘)

37 + 6 -422 NS

41 k6

GFR (mf.min-t)

3419 + 581 2281 f 5f6 0.025

1928 & 357 -2842 t_ 382 0.025

4770 f 733

REAB (flmol.min-‘)

Na+

1139 & 179

EXCR (pmo).min-‘)

3154 f 534 2378 f 510 0.025

116 f 173 2219 & 635 -2880 +402 0.025

4599 + 715

EXCR ‘IREAB (~mol.min-‘) (fimoi~min-“)

474 f 86 93 f 52 NS

381 f 48 379 It: 74 -96 lir 23 0.025

474 f 81

HCO, EXCR REAB (~mol.min-‘) (Ctmol~min-‘) 02

132 + 24 -W&30 0.025

292 + 48

Renal p02 Wnof.min’)

b.w.- ‘)

0.51 * 0.05 0.44 f 0.08

0.91 + 0.04 0.40 f 0.08

0.51 4 0.05 0.41 rt: 0.08

0.63 + 0.07 0.30 _+ 0.07 0.49 f 0.04 0.33 + 0.02 0.40 f 0.06 0.46 + 0.08

0.82 f 0.06 0.33 + 0.06

0.38 & 0.08 0.46 + 0.08

0.53 + 0.9 0.40 f 0.10

0.54 * 0.09 0.41 t 0.10 0.87 & 0.05 0.36 f 0.08

0.69 + 0.12 0.31 + 0.07

Iodide

0.88 f 0.05 0.35 f 0.10

Chloride

0.92 f 0.04 0.38 f 0.10

Bromide

Values are mean + SE of experiments in six dogs in each of the two groups.

EIC

c---E -

Group If Control (C) Ethacrynic acid (E) (5mr.kg b.w.-‘)

~~g~kg WC

Group I Control (C) Ouabain (0)

Thiocyanate

Table 2. Fractional reabsorption of anions

Mean rt SE of experiments in six dogs. Abbreviations: MABP = mean aortic blood pressure: RBF = renal blood flow: GFR = gfomerular filtration rate; EXCR = excretion: REAB = reabsorption; Renal VOL = renal oxygen consumption; P = probability.

135 f I

Acetazolamide (1OOmg~kg b.w.-‘)

(mm Hg)

MABP

Table 1. Etfect of ouabain on sodium reabsorption and oxygen consumption during acetazofamide infusion

z

B0

?J 4 B

-I g Is 3 E F z

248

FREDRIK KIIL, OLE M. SUERSTED and PETTERA. STEEN

anions. As shown in the upper part of the table, ouabain reduced fractional reabsorption of bromide and chloride to the same extent, whereas the fractional reabsorption of iodine was less reduced. The lower part of Table 2 shows that ethacrynic acid inhibited bromide and thiocyanate reabsorption as much as chloride reabsorption, whereas the inhibitory effect on iodine reabsorption was small. DISCUSSION

By reducing sodium reabsorption and renal oxygen consumption with ouabain a ANa/AOz ratio of 17.8 k 1.5 was obtained. This ratio suggests that the transport across the peritubular cell membrane by Na-K-ATPase of three moles sodium ions for each mol ATP hydrolyzed, is the only energy-requiring step in the transcellular reabsorption of NaCl and that accordingly the transport of NaCI across the luminal cell membrane does not require additional energy. In our study, bicarbonate reabsorption was also slightly reduced. Transcellular reabsorption of sodium requires the same energy whether chloride or bicarbonate are the accompanying anions (Kiil et al., 1979). However, if NaHCO, reabsorption in the proximal tubules is inhibited by ouabain, the bicarbonate dependent osmotic reabsorption of water and NaCl would also be inhibited. If this were true, the ANa/AO* ratio would be slightly higher, and an energy-requiring transport across the luminal cell membrane more probable. However, whereas l/18 mol of oxygen is consumed for the transport of 1 mol sodium by Na-K-ATPase, our data suggest that the transport across the luminal cell membrane would require less than 1/2OOmol oxygen per mol sodium transported. This negligible quantity includes all other energy requiring processes which may be reduced by ouabain administration. Before concluding that transport of NaCl across the luminal cell membrane does not require energy, factors influencing ANa/AOr are discussed. The ANa/AO* ratio is independent of the degree of inhibition. In fact ouabain may stimulate sodium reabsorption by some Na-K-ATPase molecules. By incomplete inhibition, the sodium concentration on the inside of the basolateral membrane would rise and stimulate the activity of the remaining intact molecules. In vitro studies suggest that first at concentrations above 5&60 mmol.l- ’ no further stimulation would be obtained by increasing intracellular sodium concentration. Inhibition of sodium reabsorption by ouabain implies therefore, that intracellular sodium concentration is greatly raised. In our study the transcellular NaHCOs reabsorption in the proximal tubules probably was not inhibited at all. The main reason is that the cellular uptake of sodium by the Na+/H+ exchange system in the luminal cell membrane may be stimulated by increased supply of NaHCO,, but not by increasing the supply of NaCl (Kiil, 1978). More than 60% of the total amount of Na-K-ATPase is localized in the proximal tubules of dogs (Sejersted et a/., 1979). The fractional sodium reabsorption by the bicarbonate dependent system may also be about 60x, but no more than one of three sodium ions are reabsorbed through the cells (Mathisen et al., 1979b). Hence,

there is a great surplus of Na-K-ATPase for sodium transport by the bicarbonate dependent system. Moreover, administration of acetazolamide inhibits NaHCO, reabsorption and would therefore further reduce the intracellular sodium concentration at the basolateral cell membrane. Inhibition of the bicarbonate dependent sodium transport by ouabain is therefore unlikely, whereas increased delivery of bicarbonate may raise NaHC03 reabsorption in the distal and intracellular sodium concentration nephron, approaches the saturation level for Na-K-ATPase activity. Another complication which may influence ANa/A02 determinations, is the vasoconstrictive effect of ouabain; although it rapidly diminishes, it does not completely disappear. In many studies the inhibitory effect of ouabain on sodium reabsorption has been small because of severe initial vasoconstriction. We solved this problem by infusing ouabain during intrarenal acetylcholine infusion so that the renal vessels are dilated (Lie et al., 1974). Any effect of acetylcholine disappears when the infusion is stopped, whereas the effect of ouabain lasts for hours in dogs. Although RBF recovers, GFR may be more permanently reduced, suggesting persisting intrarenal vasoconstriction. The vasoconstrictive effect may not be evenly divided between all nephrons. Nephron heterogeneity may accordingly be evoked by ouabain. Increased nephron heterogeneity would, however, have no consequence for the interpretation of the ANa/AOz ratio. In nephrons with reduced GFR, delivery of NaCl to the diluting segment would be small, whereas it would be high in super-perfused nephrons. Because of the direct proportionality between sodium transport and energy requirement, nephron heterogeneity would not alter the ANa/AO, ratio. For the same reason, the ANa/A02 ratio is not dependent on whether ouabain inhibits transcellular NaCl reabsorption completely or incompletely. The assumption that the oxygen consumption can be taken as an index of ATP generation requires some qualifications. Many naturally occuring substances, such as glycine, glucagon and dopamine, may stimulate renal energy metabolism unrelated to sodium reabsorption (Johannesen et al., 1976, 1977). Elevation of plasma concentration of fatty acids increases oxygen consumption of the heart without increasing its mechanical performance but we have, in an unpublished study, found no evidence of similar effects on renal metabolism. A more important problem is whether anaerobic metabolism is stimulated. A translation from ANa/AO* to ANa/AATP would be impossible if the diluting segment had significant anaerobic metabolism. In our study haematocrit was reduced by bleeding two days prior to the experiment to ensure reliable measurements of renal oxygen consumption. By this procedure GFR and RBF are not reduced because RBF is much larger than necessary for nutritional purposes. There is no release of lactate, as may be encountered when RBF is greatly reduced by constricting the renal artery. Ouabain infusion reduces lactate consumption to insignificant levels. Net release of lactate may increase, although anaerobic metabolism is unlikely. Even if lactate release was the consequence of anaerobic metabolism, the maximal error

249

Transceliular NaCl transport

nate dependent reabsorption of water and NaCl does due to lactate production would be 3-4x in the deternot vary during variations in GFR over a wide range. mination of the ANa/A02 ratio. Under conditions of inadequate oxygen supply, as To examine the energy requirement of transcellular NaCl reabsorption, ouabain (9Opg.kg b.w.-‘) was in isolated preparations perfused with oxygenated injected into the renal artery of anaesthetized dogs Ringer solutions, com~tition between sodium transOuabain injection port and other energy-requiring processes might well after acetazolamide administration. and oxygen consumpexist. Under such conditions the limited supply of halved sodium reabsorption tion. The ratio between the changes, 17.8 k 1.5, is not oxygen would become available for other purposes different from the ratio expected for Na-K-ATPase after reduction of sodium reabsorption. Consequently, mediated sodium transport. Bromide and thiocyanate a spuriously high ANa/AOz ratio would be obtained reabsorption was inhibited as extensively as chloride by ouabain inhibition. Our study showed that the facilitated transport of reabsorption by ouabain or ethacrynic acid. Thus. NaCl across the luminal cell membrane is not specific Na-K-ATPase transport across the basolateral memacross the luminal cell for chloride, since ouabain or ethacrynic acid in- brane facilitates transport hibited reabsorption of bromide and thiocyanate as membrane of sodium together with chloride and much as chloride reabsorption. Facilitated co- other permeable ions without additional energy retransport across the luminal cell membrane may be quirement. energized by the electrochemical potential created by Acknowieriyements-The skilled technical assistance of the Na-K-ATPase activity in the basolateral cell membrane. Such a mechanism was postulated by Miss Ann Kristin Andresen, Mrs Ashild Salvesen, Mrs Frizzell rf al. (1975) for transport of NaCl into the Grethe Laerum, Mrs Anna Croucher and Ove Moen is cells of rabbit gallbladders. One condition for such a gratefully acknowledged. This study was supported by the Norwegian Research postulate is that the ratio between extra and intraCouncil for Science and the Humanities, the Norwegian cellular concentrations is higher for chloride than for Council on Cardiovascular Diseases, Anders Jahre’s Fund sodium. In their preparation, intracellular concenfor the Promotion of Science, and Professor Carl SembS trations averaged 66 f 3 mmol.l-’ for sodium and Medical Research Fund. 84 & 3 mmol*l-’ for chloride. The ratio between intracellular chloride and sodium concentrations is probably even higher in the diluting segment, but REFERENCES intracellular measurements during high rates of NaCl BONTING S. L. & CARAVAGGIO L. L. (1963) Studies on reabsorption are not available. sodium-pot~sium-activated adenosinetriphosphat~eA second condition for co-transport of sodium and V. Correlation of enzyme activity with cation flux in six chloride ions is, that no additional energy is required. tissues. Archs Biochem. Biophys. 101, 3746. Martin & Diamond (1966) examined the energy FRIZZELLR. A., DUGAS M. C. & SCHULTZS. G. (1975) metabolism of the rabbit gallbladder and found that Sodium chloride transport by rabbit gallbladder. J. gen. only 9% of the oxygen uptake of the isolated gallPhysiol. 65, 169-795. bladder seems to be used for NaCl transport, and that HAINLINEA. (1958) Hemoglobin In Standard Merhods oj the ANa/AOZ ratio is 25. As already pointed out, a CIiniruf Chemistrv. 1st edn, Vol. 2. DD. . 49-68. Academic spuriously high ANa/AO, ratio would be obtained if Press, New York: JOHANNESEN J., LIE M. & KIIL F. (1977) Effect of glycine there is a lack of substrate or oxygen. No conclusions and gIucagon on glomerular filtration and renal metacan therefore confidently be drawn from these data bolic rates. Am. .I. Physiol. 233,‘F61-F66. concerning the energetics of coupled NaCl transport. J., LIE M., MATHISEN0. and KIIL F. (1976) These objections do not apply to the present study. JOHANNESEN Dopamine-induced dissociation between renal metabolic We conclude that NaCl transport across the luminal rate and sodium reabsorption. Am. J. Physiol. 230, cell membrane is energized by the Na-K-ATPase 1126-1131. mediated transport of sodium across the peritubular Km_ F., MONCLA~RT. & MATHISEN0. (1979) Energy recell membrane; the coupled NaCl transport across quirement for sodium reabsorption in the proximal the luminal cell membrane is not specific for chloride. tubules. Upsala J. med. Sci. Supplement 26, 38. Thus, during saline loading the Na-K-ATPase KIIL F., AUKLANDK. & REFSUMH. E. (1961) Renal sodium transport and oxygen consumption. Am. .I. Physiol. 201, mediated transport across the peritubular cell mem511-516. brane is the rate limiting step for transcellular NaCl KIIL F. (1978) Principles of active sodium reabsorption in reabsorption, whereas the bicarbonate dependent the kidney. Stand. J. clin. Lab. Invest. 38, 597402. osmotic NaCl reabsorption in the proximal tubules is KJEKSHUSJ., AUKLANDK. & KIIL F. (1969) Oxygen cost of not affected. Measurements of heat production in the sodium reabsorption in proximal and distal parts of the cortex and outer medulla support this conclusion. nephron. Scond. J. clin. Lab. Invest. 23, 307-316. The maximal capacity for transcellular NaCl reab- LIE M., SFJERSTED0. M., RAEDERM. & Knr F. (1974) sorption is reached when 1520% of the filtered load Comparisons of renal responses to ouabain and ethacrynit acid. Am. J. Physiol. 226, 1221-1226. is excreted (Monclair et al., 1978). The capacity is MARTIN D. W. & DIAMONDJ. M. (1966) Energetics of greatly reduced at plasma potassium concentrations coupled active transport of sodium and chloride. J. gee. below 2 mmol*I-” whereas hy~k~emia as well as Physiof. 50, 295-3 15. ouabain are without effect on the bicarbonate depenMATHISEN0., HOLDAASH. & KIIL F. (1979a) Scan& J. c/in. dent water and NaCl reabsorption (Monclair et al., Lab. Invest. 39, 297-301. 1980). MATH~SEN 0., MONCLAIRT., RAEDERM. & KIIL F. (1979b) 1

SUMMARY

During acetazolamide

infusion

proximal bicarbo-

Coupling of NaHCO, and NaCl reabsorption in dog kidneys during changes in plasma P,,,. Am. J. Physiol. 236 (31, F232-F239.

250

FREDR~KKIIL, OLE M. SEJERSTED and PETTERA. STEEN

MONCLAIRT., MATHISEN0. & KIIL F. (1978) Characteristics of transcellular NaCl reabsorption in the kidney. Stand. 3. clin. Lab. Invest. 38, 615-626.

MONCLAIRT., SUERSTED0. M. & KIIL F. (1980) Influence of plasma potassium concentration on the capacity for sodium reabsorption in the diluting segment of the kidney. Stand. .I. clin. Lab. Invest. 40, 27-36. PE~GREW A. R. & FELL G. S. (1972) Simplified colorimetric determination of thiocyanate in biological fluids, and its application to investigation of the toxic amblyopias. Clin. Chem. 18, 996-1000. REFSUMH. E. & SVEIN~S~N S. L. (1956) Spectrophotometric determination of hemoglobin oxygen saturation in hemolyzed whole blood. Stand. .I. din. Lab. Invest. 8, 67-70.

ROLF D., SIJRTSHINA. SC WHIR H. L. (1949) A modified diphenylamine procedure for fructose or inulin determination. Proc. Sot. exp. Biol. Med. 72, 351-354.

SWERSTED 0. M., MATHISEN0. & KIIL F. (1977) Oxygen requirement of renal Na-K-ATPase-dependent sodium reabsorption. Am. J. Physiol. 232, (2), Fl52-Fl58. SWERSTED0. M., HOLDAASH. & MONCLAIR T. (1978) Functional differences of ouabain and ethacrynic acid on renal potassium metabolism in dogs. Stand. J. c/in. Lab. Invest. 38, 603-614.

SUERSTED0. M., NICQLAUENA., MONCLAIRT. & N~coLAYSENG. (1979) Distribution of ouabain binding sites along the dog nephron. Acta physiol. stand. In press. SEN A. K. & POST R. L. (1964) Stoichiometry and locaiization of adenosine triphosphate dependent sodium and potassium transport in the erythrocyte. .I. biol. Chem. 239, 345-353. SMITHH. W., FINKELSTEIN N., ALIMINOSA A. L., CRAWFORD B. & GRABERM. (1945) The renal clearances of substituted hippuric acid derivatives and other aromatic acids in dog and man J. c/in. Invest. 24, 388404.