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Characterization of renal tubular transport of sodium chloride and water as studied in single nephrons
The American Journal JANUARY
of Medicine
1963
Edi tori al Characterization
of Renal Tubular Transport
of Sodium Chloride and Water as Studied in Single Nephrons” renal tubules constitute an epithelial system across which there is ion transfer that can be studied in some detail by direct measurement of net movement and concentration gradients of ions at chosen tubular sites. Measurement of the electrical potential difference across the tubular wall also is possible, and has aided in defining the physical forces acting on ions as they move across the tubular epithelial boundary. Such information, not easily obtained from any other type of renal function studies, permits one to characterize the sites, magnitude and driving forces involved in the process of renal tubular transport of sodium chloride and water. The renal tubular epithelium shares the characteristics of numerous other epithelial structures: it consists of electrically asymmetrical cells. Recent studies have provided evidence that the membranes bounding the luminal and peritubular surfaces of the cell have different properties, and that net transport of various species of ions results as a consequence of a typical distribution of the processes of selective diffusion and active transport at the two tubular cell membranes. In the following discussion, experiments on single nephrons will be surveyed which bear on these problems. No emphasis
T
will be placed on the renal papillary countercurrent system.
HE
Micropuncture Studies of Renal Tubular Transfer of Sodium Chloride and Water in the Mammalian Nephron. Recent micropuncture studies [ 7,2]
fully confirm earlier work [3] which showed that about 65 per cent of filtered sodium chloride and water is absorbed isosmotically from the proximal convolution, leaving further sodium chloride and water reabsorption to Henle’s loop, the distal tubule and the collecting duct. Substantial osmotic gradients can be maintained by these more distal parts of the nephron. Gottschalk [7] has observed that under a great variety of experimental conditions the prokimal tubular fluid remains isosmotic. The concentration of sodium normally remains identical with that in plasma, whereas the concentration of chloride may exceed that of the plasma significantly, implying preferential reabsorption of bicarbonate and proximal tubular acidification. Recent work on single proximal mammalian nephrons has provided direct confirmation of a’significant fall in the pH [45,22] and also has demonstrated the appearance of ammonia [6] at this tubular site. Direct evidence in support of the thesis that sodium transport in the proximal tubule is
* This work was supported by grants from the National Science Foundation and the American Heart Association. It was carried out during the tenure (G. G.) of an Established Investigatorship of the American Heart Association. 1
2
Editorial
active, and constitutes the cornerstone of water reabsorption, has come from micropuncture studies [1,2] in which the relationship between net sodium and water movement was studied during infusion of a poorly reabsorbable solute. The results of such experiments, in which marked diuresis was induced by infusion of hypertonic mannitol solution, indicate that under these conditions the net movement of water across the proximal tubule is less than under non-diuretic conditions, and that the concentration of sodium in the proximal tubular fluid falls below that of plasma. Such a concentration gradient for sodium develops because mannitol is restrained to the tubular lumen and the sodium concentration of the fluid abstracted from the tubule, osmotically equivalent to tubular fluid, is higher than that of the tubular contents. Accordingly, net movement of sodium occurs against a chemical potential gradient. Since the lumen is electronegative with respect to the peritubular fluid, the movement of sodium also occurs against an electrical potential gradient. Thus the net movement of sodium ions must be due to active transport, i.e., an energy-consuming process which accomplishes “uphill” movement of sodium ions against an electrochemical potential gradient. Such micropuncture studies have also shown that there is negligible net movement of water in the absence of movement of sodium chloride, indicating that the latter is the only significant osmotic driving force for water movement. Evaluation of tubular reabsorption of sodium when the filtered load of sodium is augmented characterization of the permits additional proximal and distal tubular systems for sodium transport, suggesting a fundamental difference in the behavior of the proximal convolution and the loop of Henle as compared to that of the more distal parts of the nephron. This conclusion is based on a study in which reabsorption of water and sodium at various tubular sites was compared in animals receiving isotonic sodium chloride solutions with that of rats given infusions of hypertonic sodium chloride solution [7]. Such micropuncture studies show that fractional reabsorption of water and sodium across the proximal tubular epithelium is not different under these conditions. Over a fractional excretion range from some 5 to 12 per cent of the filtered sodium load, sodium reabsorption in the proximal tubule increases as the sodium load is increased, due to the higher concentra-
tion of sodium in the glomerular filtrate. No maximal tubular transfer rate for sodium can be made out in the proximal tubule, and the absolute amount reabsorbed is roughly proportional to the intraluminal sodium concentration. Net sodium reabsorption increases also in the ascending part of Henle’s loop. The transfer capacity of the distal tubular epithelium, on the other hand, and that of the collecting duct system, does not similarly increase in response to loading with hypertonic sodium chloride solution. It appears to operate close to a transfer maximum (Tm) and the direct natriuretic and diuretic effect of increasing the sodium concentration in plasma seems to be exerted by surpassing this distal and collecting duct transport maximum for sodium. Microperfusion Studies of Single Nephrons Relating to the Renal Transfer of Sodium Chloride. The
method of microperfusion of single tubules, originally introduced by Richards and Walker [a], has recently been modified and extended [9] to the quantitative study of some problems of transtubular transport of water and solutes. Microperfusion of single tubular segments of mammalian nephrons also has become possible [YO], and the use of isotopes has permitted measurement of unidirectional ion movements across selected parts of the nephron [ 77,121. The particular advantage of microperfusion lies in the fact that it allows one to examine the effect of tubular activity upon a fluid sample of known composition and size independently of glomerular filtration. It also permits extensive manipulation of the composition of the test sample. Particularly useful have been “stoppedflow perfusions” in which a fluid sample is deposited between oil droplets in a tubular segment for a known period of time and subsequently withdrawn for chemical analysis. Perfusion studies in amphibian [13] and mammalian kidneys [ 101 in which various sodium chloride concentrations were used in stopped-flow perfusions, but in which the chemical activity of water was kept constant by adding mannitol, have also indicated that water transport across the proximal tubule depends upon the intratubular sodium concentration. A linear relationship was observed between the sodium chloride concentration in the lumen and the volume of water moving across the tubular epithelium. Such studies have provided additional evidence that net transport of sodium chloride can take place against conAMERICAN
JOURNAL
OF
MEDICINE
Editorial centration gradients of considerable magnitude. Independent evidence for active sodium transport, and for the thesis that the movement of water across the proximal tubular wall is secondary to the movement of sodium chloride, is thus available. If water movement were due to other forces, it would be expected to be independentoftheintratubularsodiumconcentration. The measurement of individual ion fluxes across the tubular epithelium by observing the disappearance rate of intratubularly injected isotopes is an additional means by which the kinetics of transtubular ion movement may be defined. A number of points have evolved from such studies. One is that the proximal net fluxes of sodium and chloride constitute only a relatively small fraction of the unidirectional ion movements [ 77,721. This indicates a fast turnover rate of intratubular sodium chloride, a low resistance to ion movement, and considerable back flux of sodium chloride opposite to the direction of net movement. Although such high permeabilities may, at first glance, appear to be inefficient from the point of view of energy expenditure, it should be kept in mind that they are mandatory for the osmotic coupling of solute and water transfer across the proximal tubular epithelium. Another problem which has been approached by perfusing single tubular segments with radioisotopes is that of the nature of the transfer of chloride in the proximal tubule. A quantitative assessment of this process is possible by considering the movement of chloride in relation to the chemical and electrical potential gradient involved. From such studies it appears that the degree of intratubular negativity is large enough to create an electric field of adequate strength to account for passive reabsorptive movement of chloride [ 721. No expenditure of cellular energy is necessary for the reabsorptive movement of chloride in the proximal tubule since it occurs electrochemical potential on a “downhill” gradient. Electrical Potential Dtfferences and the ShortCircuit Current Across Single Amphibian and MamKnowledge of the magnitude malian Nephrons.
and polarity of the electrical potential gradient across the renal tubular epithelium is essential for analysis of the driving forces acting on sodium and chloride ions. Since all segments of the tubular lumen have been found to be electrically negative, to a varying degree, with respect to the peritubular fluid [74-771, a nonVOL.
34,
JANUARY
1363
3
specific driving force is provided which aids in the transfer of anions out of the tubular lumen and that of cations into it. Additional evidence obtained by direct electrical measurements of single proximal kidney cells has shown that the cell interior is electrically negative with respect to both the tubular lumen and the peritubular fluid [ 74,76,78,79]. The transtubular electrical potential profile, respective ionic concentration gradients and the direction of net movement of sodium chloride have provided the basis for a cell model which allows a more detailed analysis of sodium reabsorption in the proximal tubule [ 78-201. The key factor is a sodium extrusion mechanism located at the peritubular membrane of the renal tubular cell. The activity of this metabolically driven pump maintains a low intracellular sodium concentration. * This low intracellular sodium concentration, coupled with the electronegativity of the cell interior with respect to the tubular lumen, constitutes a sink, allowing sodium ions in the tubular lumen to diffuse passively through the luminal membrane along its electrochemical potential gradient into the cell. Intracellular electronegativity is established, essentially, by the high permeability of the tubular cell to potassium ions, and a potassium concentration in the cell interior which is maintained at a level many times higher than that of the surrounding extracellular medium. Potassium diffusion potentials which develop as a consequence of the high selective permeability of the kidney cell to this ion determine the magnitude of intracellular negativity. These potential
Editorial meability at the luminal cell membrane creates a preferential pathway for positively charged sodium ions and more extensively reduces the electrical potential difference at this site. Thus the tubular cell is rendered asymmetrically negative with respect to the tubular lumen (about -50 mv.) and peritubular fluid (about -70 mv.). * Steeper electrical potential gradients have been observed across the distal tubular epithelium, a site where the administration of non-penetrant anions such as sulfate or ferrocyanide leads to a significant increase in intratubular negativity [77]. While such electrical potential gradients provide a powerful force to expel chloride ions from the lumen, a recent study by Rector and Clapp [25] has shown that it is insufficient to account for the extremely low chloride concentrations found under these experimental conditions. Accordingly, these studies indicate the existence of an active transport process for chloride at the distal tubular level. The observed increase in the transtubular potential difference may also be involved importantly in the known augmentation of potassium and hydrogen ion excretion under conditions of loading with non-penetrant anions. It should be noted that the electrical potential difference may affect the distribution of potassium and hydrogen ions in at least two different ways [ 171. If potassium and hydrogen ions were secreted into the distal tubular lumen in exchange for tubular sodium, a higher the intratubular negativity might restrain diffusion of these cations out of the tubular lumen, and thus cause enhanced net secretion. If, on the other hand, potassium and hydrogen ions are in part passively distributed between * Some additional remarks are in order. First, it should be recognized that the phenomena which led to the proposed cell schema were obtained in proximal tubule cells of Necturus, a species characterized by the large size of individual tubule cells. It is, however, likely that the same basic electrical potential profile also exists in mammalian species. However, the importance of a number of other factors in the generation of the observed electrical asymmetry across the mammalian nephron is less clear. It is possible that unequal chloride permeabilities at the luminal and peritubular cell membranes [22], active potassium uptake at the luminal cell membrane [23] and the apparent lack of electrochemical equilibrium for chloride ions across the proximal tubular wall [24 may be contributing factors. Also, the role of hydrogen ion secretion is not clear. It should be emphasized further that the existence of an electrochemical potential gradient favoring downhill movement of sodium at the luminai cell membrane does not exclude some participation of carrier-mediated sodium transfer or linked sodium-potassium exchange at this site.
tubular cell and luminal fluid, increased tubular negativity would similarly promote increased excretionzrate by electrostatic attraction into the lumen. The distinction between these two possibilities can be made only by careful correlation of flux measurements with simultaneously determined electrochemical potential gradients. Such studies and others concerned with a truly critical evaluation of the exact role of the transtubular potential difference in many tubular ionic transport processes have not yet been carried out, but it is obvious that they will have an important bearing on the future analysis of tubular ionic transport processes. The significance of the transtubular potential difference in defining the processes of tubular transport of sodium chloride has also been emphasized by recent studies concerned with the mapping of electrical gradients in the renal medulla [26]. Thus, in measurements of transtubular potential differences in the ascending limb of Henle’s loop and in the collecting ducts, the lumen was consistently found to be electrically negative with respect to Ringer’s solution in the peritoneal cavity. If sodium chloride moves out of the ascending limb of Henle’s loop, it must be concluded that such net movement constitutes movement of sodium ions against an electrochemical potential gradient. Thus it would be active also in nature. In the case of chloride, data are consistent with, but donot provide unequivocal proof of, a passive mode of transfer out of the ascending limb of Henle’s loop. The measurement of the short-circuit current, i.e., the amount of electrical charge actively transferred across the renal tubule at zero potential difference, has recently provided another electrophysiological approach for the study of tubular electrolyte transfer. * The results * Measurements of the short-circuit current [30] can be applied to situations in which the solutions on both sides of a biological membrane are identical in composition, a condition approximated in the case of the proximal tubular epithelium in both amphibian and mammalian kidneys. An electrical potential difference under such conditions is usually generated by active transport of one or several ion species. If under such conditions an external source of an electromotive force is provided, the membrane potential difference can be balanced out exactly. Under these conditions of zero potential difference across the membrane, the current which flows is, by definition, the short-circuit current. It equals the quantity of electrical charge which is transferred in the absence of either an electrical or chemical potential gradient, i.e., actively, across a biological membrane per unit time and area. AMERICAN
JOURNAL
OF
MEDICINE
Editorial of such studies on an amphibian nephron [27] may be summarized by stating that the proximal transtubular short-circuit current is in reasonable agreement with net sodium flux. Furthermore, the contribution of chloride movement to the short-circuit current is negligible, and, finally, the short-circuit current is only slightly decreased in the absence of all colloids in perfused kidney preparations, i.e., in the absence of a colloid-osmotic pressure gradient across the proximal tubular epithelium. Comparison of the short-circuit current with calculated sodium movement in perfusion experiments of singIe proximal tubules indicates again that in the rat [70] a major fraction of sodium reabsorption is active in nature. The observation that the current that can be drawn from single segments of the renal tubular epithelium approximates or exceeds net movement of sodium argues against an important role of the colloid osmotic pressure difference in promoting reabsorption of proximal tubular fluid and electrolytes. GERHARD
GIEBISCH,
M.D.
Department of Physiology Cornell University Medical College New York, New York ERICH
AND
E. WINDHAGER,
M.D.
Institute of Biological Chemistry University of CojWnhagen Copenhagen, Denmark
REFERENCES C. W. Micropuncture studies of tubular function in the mammalian kidney. The
1. G~TISCHALK,
Physiologist, 4: 35, 1961. 2. WINDHA~ER, E. E. and GIEBISCH,G. Micropuncture study of renal tubular transfer of sodium chloride in the rat. Am. J. Physiol., 200: 581, 1961. 3. WALKER, A. M., BOTT, P. A., OLTVER, J. and MACDOWELL, M. Collection and analysis of fluid from single nephrons of the mammalian kidney. Am. J. physioI.;134: 580, 1941. 4. GOTTSCHALK. C. W.. LASSITER.W. E. and MYLLE, M. Localization of urine acidification in the mam: malian kidney. Am. J. Physiol., 198: 581, 1960. 5. GIEBI~CH,G., WINDHAGER, E. E. and Pnm, R. F. Mechanism of urinary acidification. In: Biology of Pyelonephritis, p. 277. Edited by Quinn, E. L. and Kass, E. M. Boston, 1960. Little, Brown & co. 6. GLABMAN, S. and GIEBIXH, G. Unpublished observations. 7. GIEB~XH, G., KLOSE, R. and WINDHAGER, E. E. Micropuncture study of renal tubular transfer of sodium and water during sodium chloride loading. Fed. Proc., 21: 432, 1961. VOL.
34,
JANUARY
1963
5
8. RICHARDS, A. N. and WALXER, A. M. Methods of collecting fluid from known regions of the renal tubules of amphibia and of perfusing the lumen of a single tubule. Am. J. Physiol., 118: 111, 1937. 9. SHIPP, J. C.. HANENSON.I. B.. WINDHAGER. E. E.. SC&ATZMA~N, H. J., ‘WHI~~~MBURY, G.,‘Yo& MURA, H. and SOLOMON, A. K. Single proximal tubules of Necturus. I. Method for micropuncture and microperfusion. Am. J. Physiol., 195: 563,1958. 10. WINDHAGER,E. E. and GIEBISCH,G. Comparison of short-circuit current and net water movement in single perfused proximal tubules of rat kidneys. Nature, London, 191: 4794, 1961. 11. OKEN, D. E., WHITTEMBURY, G., WINDHAGER, E. E., SCHATZMANN,H. J. and SOLOMON, A. K. Active sodium transport by the proximal tubule of Necturus. J. Clin. Invest.. 38: 1029. 1959. 12. GIEBI~CH,G. and WINDHAGER,E. E. ‘Chloride fluxes across single proximal tubules of Necturus kidney. Fed. Proc., 18: 52, 1959. 13. WINDHAGER,E. E., WHITTEMBURY,G., OKEN, D. E., SCHATZMANN,H. J. and SOLOMON,A. K. Single proximal tubules of Necturus kidney. III. Dependence of water movement on sodium chloride concentration. Am. J. Physiol., 197: 313, 1959. 14. GIEBISCH,G. Electrical potential measurements on single nephrons of Necturus. J. CeN. B Corn@ Physiol., 51: 221, 1958. 15. SOLOMON, S. Transtubular potential differences of rat kidney. J. Cell. B Camp. Physiol., 49: 351, 1957. 16. WHITTEMBURY,G. and WINDHAGER,E. E. Electrical potential difference measurements in perfused single proximal tubules of Necturus kidney. J. Gen. Physiol., 44: 679, 1961. 17. CLAPP, J. R., RECTOR, F. C. and SELDIN,D. Effect of unreabsorbed anions on proximal and distal transtubular potentials in rats. Am. J. Physiol., 202: 781, 1962. 18. GIEBISCH, G. Measurements of electrical potential differences on single nephrons of the perfused Necturus kidney. J. Gen. Physiol., 44: 659, 1961. 19. WHI~MBURY, G., SUGINO,N. and SOLOMON,A. K. Ionic permeability and electrical potential differences in Necturus kidney cells. J. Gen. Physiol., 44: 687, 1961. 20. GIEBISCH,G. Measurements of electrical potentials and ion fluxes on single renal tubules. Circulation, 21: 879, 1960. 21. KLOSE, R. M., GIEBISCH,G. and WINDHAGER,E. E. Unpublished observations. between electrical and 22. BANK, N. Relationship hydrogen ion gradients across rat proximal tubule. Am. J. Pf+rioL, 203: 577, 1962. 23. MARSH, D. and RUMRICH,G. Micropuncture analysis of potassium concentrations in mammalian renal tubular fluid. In: Proceedings of International Union of Physiological Sciences, XXII International Congress, vol. II, p. 251. Leiden, 1962. 24. GERTZ, K. H. Direct measurement of the transtubular flux of electrolytes and non-electrolytes in the intact rat kidney. In: Proceedings of International Union of Physiological Sciences, XXII International Congress, vol. x, Symposium VII, p. 370. Leiden, 1962.
Editorial 25. RECTOR, F. C., JR. and CLAPP, J. R. Evidence for active chloride reabsorption in the distal renal tubule of the rat. J. Clin. Inuest., 41: 101, 1962. 26. WINDHAGER,E. E. Evidence for active transport of sodium in loops of Henle. In: Proceedings of International Union of Physiological Sciences, XXII International Congress, vol. II, p. 252. Leiden, 1962. 27. EIGLER, F. W. Short-circuit current measurements in proximal tubule of Necturus kidney. Am. J. Physiol., 201: 157, 1961.
28. SCHATZMANN, H. J., WINDHAGER, E. E. and SOLOMON,A. K. Single proximal tubules of the Necturus kidney. II. Effect of 2,4 dinitrophenol and ouabain on water reabsorption. Am. J. Physiol., 195: 570, 1958. 29. ORLOFF, J. and BURG, M. Effect of strophantidhin on electrolyte excretion in the chicken. Am. J. Physiol., 199: 49, 1960. 30. USSING, H. H. In: The Alkali Metal Ions in Biology, Handbuch der experimentellen Pharmakologie. Edited by Eichler, 0. and Farah, A. Berlin, Goettingen, Heidelberg, 1960. Springer Verlag.
AMERICAN
JOURNAL
OF MEDICINE
of Medicine
1963
Edi tori al Characterization
of Renal Tubular Transport
of Sodium Chloride and Water as Studied in Single Nephrons” renal tubules constitute an epithelial system across which there is ion transfer that can be studied in some detail by direct measurement of net movement and concentration gradients of ions at chosen tubular sites. Measurement of the electrical potential difference across the tubular wall also is possible, and has aided in defining the physical forces acting on ions as they move across the tubular epithelial boundary. Such information, not easily obtained from any other type of renal function studies, permits one to characterize the sites, magnitude and driving forces involved in the process of renal tubular transport of sodium chloride and water. The renal tubular epithelium shares the characteristics of numerous other epithelial structures: it consists of electrically asymmetrical cells. Recent studies have provided evidence that the membranes bounding the luminal and peritubular surfaces of the cell have different properties, and that net transport of various species of ions results as a consequence of a typical distribution of the processes of selective diffusion and active transport at the two tubular cell membranes. In the following discussion, experiments on single nephrons will be surveyed which bear on these problems. No emphasis
T
will be placed on the renal papillary countercurrent system.
HE
Micropuncture Studies of Renal Tubular Transfer of Sodium Chloride and Water in the Mammalian Nephron. Recent micropuncture studies [ 7,2]
fully confirm earlier work [3] which showed that about 65 per cent of filtered sodium chloride and water is absorbed isosmotically from the proximal convolution, leaving further sodium chloride and water reabsorption to Henle’s loop, the distal tubule and the collecting duct. Substantial osmotic gradients can be maintained by these more distal parts of the nephron. Gottschalk [7] has observed that under a great variety of experimental conditions the prokimal tubular fluid remains isosmotic. The concentration of sodium normally remains identical with that in plasma, whereas the concentration of chloride may exceed that of the plasma significantly, implying preferential reabsorption of bicarbonate and proximal tubular acidification. Recent work on single proximal mammalian nephrons has provided direct confirmation of a’significant fall in the pH [45,22] and also has demonstrated the appearance of ammonia [6] at this tubular site. Direct evidence in support of the thesis that sodium transport in the proximal tubule is
* This work was supported by grants from the National Science Foundation and the American Heart Association. It was carried out during the tenure (G. G.) of an Established Investigatorship of the American Heart Association. 1
2
Editorial
active, and constitutes the cornerstone of water reabsorption, has come from micropuncture studies [1,2] in which the relationship between net sodium and water movement was studied during infusion of a poorly reabsorbable solute. The results of such experiments, in which marked diuresis was induced by infusion of hypertonic mannitol solution, indicate that under these conditions the net movement of water across the proximal tubule is less than under non-diuretic conditions, and that the concentration of sodium in the proximal tubular fluid falls below that of plasma. Such a concentration gradient for sodium develops because mannitol is restrained to the tubular lumen and the sodium concentration of the fluid abstracted from the tubule, osmotically equivalent to tubular fluid, is higher than that of the tubular contents. Accordingly, net movement of sodium occurs against a chemical potential gradient. Since the lumen is electronegative with respect to the peritubular fluid, the movement of sodium also occurs against an electrical potential gradient. Thus the net movement of sodium ions must be due to active transport, i.e., an energy-consuming process which accomplishes “uphill” movement of sodium ions against an electrochemical potential gradient. Such micropuncture studies have also shown that there is negligible net movement of water in the absence of movement of sodium chloride, indicating that the latter is the only significant osmotic driving force for water movement. Evaluation of tubular reabsorption of sodium when the filtered load of sodium is augmented characterization of the permits additional proximal and distal tubular systems for sodium transport, suggesting a fundamental difference in the behavior of the proximal convolution and the loop of Henle as compared to that of the more distal parts of the nephron. This conclusion is based on a study in which reabsorption of water and sodium at various tubular sites was compared in animals receiving isotonic sodium chloride solutions with that of rats given infusions of hypertonic sodium chloride solution [7]. Such micropuncture studies show that fractional reabsorption of water and sodium across the proximal tubular epithelium is not different under these conditions. Over a fractional excretion range from some 5 to 12 per cent of the filtered sodium load, sodium reabsorption in the proximal tubule increases as the sodium load is increased, due to the higher concentra-
tion of sodium in the glomerular filtrate. No maximal tubular transfer rate for sodium can be made out in the proximal tubule, and the absolute amount reabsorbed is roughly proportional to the intraluminal sodium concentration. Net sodium reabsorption increases also in the ascending part of Henle’s loop. The transfer capacity of the distal tubular epithelium, on the other hand, and that of the collecting duct system, does not similarly increase in response to loading with hypertonic sodium chloride solution. It appears to operate close to a transfer maximum (Tm) and the direct natriuretic and diuretic effect of increasing the sodium concentration in plasma seems to be exerted by surpassing this distal and collecting duct transport maximum for sodium. Microperfusion Studies of Single Nephrons Relating to the Renal Transfer of Sodium Chloride. The
method of microperfusion of single tubules, originally introduced by Richards and Walker [a], has recently been modified and extended [9] to the quantitative study of some problems of transtubular transport of water and solutes. Microperfusion of single tubular segments of mammalian nephrons also has become possible [YO], and the use of isotopes has permitted measurement of unidirectional ion movements across selected parts of the nephron [ 77,121. The particular advantage of microperfusion lies in the fact that it allows one to examine the effect of tubular activity upon a fluid sample of known composition and size independently of glomerular filtration. It also permits extensive manipulation of the composition of the test sample. Particularly useful have been “stoppedflow perfusions” in which a fluid sample is deposited between oil droplets in a tubular segment for a known period of time and subsequently withdrawn for chemical analysis. Perfusion studies in amphibian [13] and mammalian kidneys [ 101 in which various sodium chloride concentrations were used in stopped-flow perfusions, but in which the chemical activity of water was kept constant by adding mannitol, have also indicated that water transport across the proximal tubule depends upon the intratubular sodium concentration. A linear relationship was observed between the sodium chloride concentration in the lumen and the volume of water moving across the tubular epithelium. Such studies have provided additional evidence that net transport of sodium chloride can take place against conAMERICAN
JOURNAL
OF
MEDICINE
Editorial centration gradients of considerable magnitude. Independent evidence for active sodium transport, and for the thesis that the movement of water across the proximal tubular wall is secondary to the movement of sodium chloride, is thus available. If water movement were due to other forces, it would be expected to be independentoftheintratubularsodiumconcentration. The measurement of individual ion fluxes across the tubular epithelium by observing the disappearance rate of intratubularly injected isotopes is an additional means by which the kinetics of transtubular ion movement may be defined. A number of points have evolved from such studies. One is that the proximal net fluxes of sodium and chloride constitute only a relatively small fraction of the unidirectional ion movements [ 77,721. This indicates a fast turnover rate of intratubular sodium chloride, a low resistance to ion movement, and considerable back flux of sodium chloride opposite to the direction of net movement. Although such high permeabilities may, at first glance, appear to be inefficient from the point of view of energy expenditure, it should be kept in mind that they are mandatory for the osmotic coupling of solute and water transfer across the proximal tubular epithelium. Another problem which has been approached by perfusing single tubular segments with radioisotopes is that of the nature of the transfer of chloride in the proximal tubule. A quantitative assessment of this process is possible by considering the movement of chloride in relation to the chemical and electrical potential gradient involved. From such studies it appears that the degree of intratubular negativity is large enough to create an electric field of adequate strength to account for passive reabsorptive movement of chloride [ 721. No expenditure of cellular energy is necessary for the reabsorptive movement of chloride in the proximal tubule since it occurs electrochemical potential on a “downhill” gradient. Electrical Potential Dtfferences and the ShortCircuit Current Across Single Amphibian and MamKnowledge of the magnitude malian Nephrons.
and polarity of the electrical potential gradient across the renal tubular epithelium is essential for analysis of the driving forces acting on sodium and chloride ions. Since all segments of the tubular lumen have been found to be electrically negative, to a varying degree, with respect to the peritubular fluid [74-771, a nonVOL.
34,
JANUARY
1363
3
specific driving force is provided which aids in the transfer of anions out of the tubular lumen and that of cations into it. Additional evidence obtained by direct electrical measurements of single proximal kidney cells has shown that the cell interior is electrically negative with respect to both the tubular lumen and the peritubular fluid [ 74,76,78,79]. The transtubular electrical potential profile, respective ionic concentration gradients and the direction of net movement of sodium chloride have provided the basis for a cell model which allows a more detailed analysis of sodium reabsorption in the proximal tubule [ 78-201. The key factor is a sodium extrusion mechanism located at the peritubular membrane of the renal tubular cell. The activity of this metabolically driven pump maintains a low intracellular sodium concentration. * This low intracellular sodium concentration, coupled with the electronegativity of the cell interior with respect to the tubular lumen, constitutes a sink, allowing sodium ions in the tubular lumen to diffuse passively through the luminal membrane along its electrochemical potential gradient into the cell. Intracellular electronegativity is established, essentially, by the high permeability of the tubular cell to potassium ions, and a potassium concentration in the cell interior which is maintained at a level many times higher than that of the surrounding extracellular medium. Potassium diffusion potentials which develop as a consequence of the high selective permeability of the kidney cell to this ion determine the magnitude of intracellular negativity. These potential
Editorial meability at the luminal cell membrane creates a preferential pathway for positively charged sodium ions and more extensively reduces the electrical potential difference at this site. Thus the tubular cell is rendered asymmetrically negative with respect to the tubular lumen (about -50 mv.) and peritubular fluid (about -70 mv.). * Steeper electrical potential gradients have been observed across the distal tubular epithelium, a site where the administration of non-penetrant anions such as sulfate or ferrocyanide leads to a significant increase in intratubular negativity [77]. While such electrical potential gradients provide a powerful force to expel chloride ions from the lumen, a recent study by Rector and Clapp [25] has shown that it is insufficient to account for the extremely low chloride concentrations found under these experimental conditions. Accordingly, these studies indicate the existence of an active transport process for chloride at the distal tubular level. The observed increase in the transtubular potential difference may also be involved importantly in the known augmentation of potassium and hydrogen ion excretion under conditions of loading with non-penetrant anions. It should be noted that the electrical potential difference may affect the distribution of potassium and hydrogen ions in at least two different ways [ 171. If potassium and hydrogen ions were secreted into the distal tubular lumen in exchange for tubular sodium, a higher the intratubular negativity might restrain diffusion of these cations out of the tubular lumen, and thus cause enhanced net secretion. If, on the other hand, potassium and hydrogen ions are in part passively distributed between * Some additional remarks are in order. First, it should be recognized that the phenomena which led to the proposed cell schema were obtained in proximal tubule cells of Necturus, a species characterized by the large size of individual tubule cells. It is, however, likely that the same basic electrical potential profile also exists in mammalian species. However, the importance of a number of other factors in the generation of the observed electrical asymmetry across the mammalian nephron is less clear. It is possible that unequal chloride permeabilities at the luminal and peritubular cell membranes [22], active potassium uptake at the luminal cell membrane [23] and the apparent lack of electrochemical equilibrium for chloride ions across the proximal tubular wall [24 may be contributing factors. Also, the role of hydrogen ion secretion is not clear. It should be emphasized further that the existence of an electrochemical potential gradient favoring downhill movement of sodium at the luminai cell membrane does not exclude some participation of carrier-mediated sodium transfer or linked sodium-potassium exchange at this site.
tubular cell and luminal fluid, increased tubular negativity would similarly promote increased excretionzrate by electrostatic attraction into the lumen. The distinction between these two possibilities can be made only by careful correlation of flux measurements with simultaneously determined electrochemical potential gradients. Such studies and others concerned with a truly critical evaluation of the exact role of the transtubular potential difference in many tubular ionic transport processes have not yet been carried out, but it is obvious that they will have an important bearing on the future analysis of tubular ionic transport processes. The significance of the transtubular potential difference in defining the processes of tubular transport of sodium chloride has also been emphasized by recent studies concerned with the mapping of electrical gradients in the renal medulla [26]. Thus, in measurements of transtubular potential differences in the ascending limb of Henle’s loop and in the collecting ducts, the lumen was consistently found to be electrically negative with respect to Ringer’s solution in the peritoneal cavity. If sodium chloride moves out of the ascending limb of Henle’s loop, it must be concluded that such net movement constitutes movement of sodium ions against an electrochemical potential gradient. Thus it would be active also in nature. In the case of chloride, data are consistent with, but donot provide unequivocal proof of, a passive mode of transfer out of the ascending limb of Henle’s loop. The measurement of the short-circuit current, i.e., the amount of electrical charge actively transferred across the renal tubule at zero potential difference, has recently provided another electrophysiological approach for the study of tubular electrolyte transfer. * The results * Measurements of the short-circuit current [30] can be applied to situations in which the solutions on both sides of a biological membrane are identical in composition, a condition approximated in the case of the proximal tubular epithelium in both amphibian and mammalian kidneys. An electrical potential difference under such conditions is usually generated by active transport of one or several ion species. If under such conditions an external source of an electromotive force is provided, the membrane potential difference can be balanced out exactly. Under these conditions of zero potential difference across the membrane, the current which flows is, by definition, the short-circuit current. It equals the quantity of electrical charge which is transferred in the absence of either an electrical or chemical potential gradient, i.e., actively, across a biological membrane per unit time and area. AMERICAN
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Editorial of such studies on an amphibian nephron [27] may be summarized by stating that the proximal transtubular short-circuit current is in reasonable agreement with net sodium flux. Furthermore, the contribution of chloride movement to the short-circuit current is negligible, and, finally, the short-circuit current is only slightly decreased in the absence of all colloids in perfused kidney preparations, i.e., in the absence of a colloid-osmotic pressure gradient across the proximal tubular epithelium. Comparison of the short-circuit current with calculated sodium movement in perfusion experiments of singIe proximal tubules indicates again that in the rat [70] a major fraction of sodium reabsorption is active in nature. The observation that the current that can be drawn from single segments of the renal tubular epithelium approximates or exceeds net movement of sodium argues against an important role of the colloid osmotic pressure difference in promoting reabsorption of proximal tubular fluid and electrolytes. GERHARD
GIEBISCH,
M.D.
Department of Physiology Cornell University Medical College New York, New York ERICH
AND
E. WINDHAGER,
M.D.
Institute of Biological Chemistry University of CojWnhagen Copenhagen, Denmark
REFERENCES C. W. Micropuncture studies of tubular function in the mammalian kidney. The
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Physiologist, 4: 35, 1961. 2. WINDHA~ER, E. E. and GIEBISCH,G. Micropuncture study of renal tubular transfer of sodium chloride in the rat. Am. J. Physiol., 200: 581, 1961. 3. WALKER, A. M., BOTT, P. A., OLTVER, J. and MACDOWELL, M. Collection and analysis of fluid from single nephrons of the mammalian kidney. Am. J. physioI.;134: 580, 1941. 4. GOTTSCHALK. C. W.. LASSITER.W. E. and MYLLE, M. Localization of urine acidification in the mam: malian kidney. Am. J. Physiol., 198: 581, 1960. 5. GIEBI~CH,G., WINDHAGER, E. E. and Pnm, R. F. Mechanism of urinary acidification. In: Biology of Pyelonephritis, p. 277. Edited by Quinn, E. L. and Kass, E. M. Boston, 1960. Little, Brown & co. 6. GLABMAN, S. and GIEBIXH, G. Unpublished observations. 7. GIEB~XH, G., KLOSE, R. and WINDHAGER, E. E. Micropuncture study of renal tubular transfer of sodium and water during sodium chloride loading. Fed. Proc., 21: 432, 1961. VOL.
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8. RICHARDS, A. N. and WALXER, A. M. Methods of collecting fluid from known regions of the renal tubules of amphibia and of perfusing the lumen of a single tubule. Am. J. Physiol., 118: 111, 1937. 9. SHIPP, J. C.. HANENSON.I. B.. WINDHAGER. E. E.. SC&ATZMA~N, H. J., ‘WHI~~~MBURY, G.,‘Yo& MURA, H. and SOLOMON, A. K. Single proximal tubules of Necturus. I. Method for micropuncture and microperfusion. Am. J. Physiol., 195: 563,1958. 10. WINDHAGER,E. E. and GIEBISCH,G. Comparison of short-circuit current and net water movement in single perfused proximal tubules of rat kidneys. Nature, London, 191: 4794, 1961. 11. OKEN, D. E., WHITTEMBURY, G., WINDHAGER, E. E., SCHATZMANN,H. J. and SOLOMON, A. K. Active sodium transport by the proximal tubule of Necturus. J. Clin. Invest.. 38: 1029. 1959. 12. GIEBI~CH,G. and WINDHAGER,E. E. ‘Chloride fluxes across single proximal tubules of Necturus kidney. Fed. Proc., 18: 52, 1959. 13. WINDHAGER,E. E., WHITTEMBURY,G., OKEN, D. E., SCHATZMANN,H. J. and SOLOMON,A. K. Single proximal tubules of Necturus kidney. III. Dependence of water movement on sodium chloride concentration. Am. J. Physiol., 197: 313, 1959. 14. GIEBISCH,G. Electrical potential measurements on single nephrons of Necturus. J. CeN. B Corn@ Physiol., 51: 221, 1958. 15. SOLOMON, S. Transtubular potential differences of rat kidney. J. Cell. B Camp. Physiol., 49: 351, 1957. 16. WHITTEMBURY,G. and WINDHAGER,E. E. Electrical potential difference measurements in perfused single proximal tubules of Necturus kidney. J. Gen. Physiol., 44: 679, 1961. 17. CLAPP, J. R., RECTOR, F. C. and SELDIN,D. Effect of unreabsorbed anions on proximal and distal transtubular potentials in rats. Am. J. Physiol., 202: 781, 1962. 18. GIEBISCH, G. Measurements of electrical potential differences on single nephrons of the perfused Necturus kidney. J. Gen. Physiol., 44: 659, 1961. 19. WHI~MBURY, G., SUGINO,N. and SOLOMON,A. K. Ionic permeability and electrical potential differences in Necturus kidney cells. J. Gen. Physiol., 44: 687, 1961. 20. GIEBISCH,G. Measurements of electrical potentials and ion fluxes on single renal tubules. Circulation, 21: 879, 1960. 21. KLOSE, R. M., GIEBISCH,G. and WINDHAGER,E. E. Unpublished observations. between electrical and 22. BANK, N. Relationship hydrogen ion gradients across rat proximal tubule. Am. J. Pf+rioL, 203: 577, 1962. 23. MARSH, D. and RUMRICH,G. Micropuncture analysis of potassium concentrations in mammalian renal tubular fluid. In: Proceedings of International Union of Physiological Sciences, XXII International Congress, vol. II, p. 251. Leiden, 1962. 24. GERTZ, K. H. Direct measurement of the transtubular flux of electrolytes and non-electrolytes in the intact rat kidney. In: Proceedings of International Union of Physiological Sciences, XXII International Congress, vol. x, Symposium VII, p. 370. Leiden, 1962.
Editorial 25. RECTOR, F. C., JR. and CLAPP, J. R. Evidence for active chloride reabsorption in the distal renal tubule of the rat. J. Clin. Inuest., 41: 101, 1962. 26. WINDHAGER,E. E. Evidence for active transport of sodium in loops of Henle. In: Proceedings of International Union of Physiological Sciences, XXII International Congress, vol. II, p. 252. Leiden, 1962. 27. EIGLER, F. W. Short-circuit current measurements in proximal tubule of Necturus kidney. Am. J. Physiol., 201: 157, 1961.
28. SCHATZMANN, H. J., WINDHAGER, E. E. and SOLOMON,A. K. Single proximal tubules of the Necturus kidney. II. Effect of 2,4 dinitrophenol and ouabain on water reabsorption. Am. J. Physiol., 195: 570, 1958. 29. ORLOFF, J. and BURG, M. Effect of strophantidhin on electrolyte excretion in the chicken. Am. J. Physiol., 199: 49, 1960. 30. USSING, H. H. In: The Alkali Metal Ions in Biology, Handbuch der experimentellen Pharmakologie. Edited by Eichler, 0. and Farah, A. Berlin, Goettingen, Heidelberg, 1960. Springer Verlag.
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