Sodium-free fluid reabsorption in Necturus kidney perfused with sodium-free media

Kidney International, Vol. 4 (1973). p. 252—258

Sodium-free fluid reabsorption in Necturus kidney perfused with sodium-free media DOMINIQUE VILLEY and TAKIS ANAGNOSTOPOULOS Laboratofre de Physiologie Rénale, inserm u. 30, Hôpital Necker Enfants-Malades, Paris, France

Sodium-free fluid reabsorption in Necturus kidney perfused with sodium-free media. The ability of tubular epithelium to transport sodium-free fluid from lumen to the peritubular side

suggCrent qu'un transport transCpithélial net, consommant de l'Cnergie, peut avoir lieu dans le rein de Necturus perfuse avec des solutions dCpourvues de sodium.

was tested in the isolated Necturus kidney perfused in situ with sodium-free solutions. Each animal was perfused with a single

solution. Perfusion rates were 0.11 mI/mm (portal vein) and 0.21 mI/mm (aorta). The standard Na solutions were a Ringer's bicarbonate and a Ringer's phosphate (Na, 95 mM; total osmo-

lality, 200 mOsm). The substituted solutions were of similar composition to the control Ringer's phosphate in all but the Na content. Sodium was substituted by either lithium, potassium, rubidium, cesium or choline. Large split-drops were introduced into in the proximal tubule with an aliquot of the perfusing solution. In most experiments volume reduction was actually measured until at least one-half of the split-drop was reabsorbed. It

It has been generally accepted that active transport of sodium at the peritubular border of kidney cells provides the driving force for fluid movement in proximal tubules of both amphibians [1—4] and mammals [5—7]. The usual interpretation of electrochemical gradients between cells

and surrounding media [8, 9] has been applied to the

was found that fluid transport proceeds in Na-free solutions, without important changes in reabsorptive rate. When ouabain was added to sodium and choline solutions, reabsorption was

sharply reduced. The data suggest that transepithelial net movement, metabolically dependent, may occur in Necturus kidney perfused with Na-free media.

Reabsorption tubulaire dans le rein de Necturus, perfuse avec des solutions dépourvues de sodium. La capacité de l'épithClium

tubulaire de réabsorber le fluide intra-tubulaire de Ia lumiCre vers l'interstitium a Cté étudiée dans le rein isolé de Necturus,

tubular membranes of the kidney, and the experimental data have been fitted to current concepts [1, 4]. The concept of active transport, however, implies a quantitative assessment of the magnitude of passive transport. Experiments involving compartmental studies [10—13], cation-sensitive electrode measurements [14—16] and nuclear

magnetic resonance data [17—20] suggest that a proportion of each intracellular ion might be in a different state from

that in free solution. On the other hand, the peculiarities of the solvent properties of water in complex systems [21—23] and living tissues [17—19, 23—29] are still a matter

perfuse avec diverses solutions dCpourvues de sodium. Chaque animal a Cté perfuse avec une seule solution. Les debits de perfusion Ctaient de 0.11 mI/mm (veine porte) et 0.21 mI/mm

of extensive controversy.

The implications of such considerations are of great

(aorte). Les solutions standard avec sodium étaient un Ringer bicarbonate et un Ringer phosphate (Na, 95 mM; osmolalité totale, 200 mOsm). Les solutions de substitution avaient la mCme composition que Ic Ringer phosphate, a l'exception du sodium qui a etC remplace par du lithium, du potassium, du rubidium, du cesium ou de Ia choline. Un aliquot de Ia solution de perfusion a été depose entre deux colonnes d'huile dans Ia lumiCre tubulaire proximate. Dans Ia plupart des cas, Ia réduction du volume de Ia gouttelette a etC observée jusqu'à rCabsorption de plus de sa moitiC. Le transport se poursuit en l'absence de sodium, sans modification majeure de la vitesse de rCabsorp-

tion. L'addition d'ouabaine dans les solutions de sodium et de choline rCduit considCrablement la reabsorption. Ces rCsultats Received for publication January 11, 1973; and in revised form April 11, 1973. © 1973, by the International Society of Nephrology.

importance in studies on transtubular reabsorptive mechanisms, since it has been assumed that the cell may be

regarded as a single compartment and that the laws of dilute solutions may be applied, at least in rough approxima-

tions. In Necturus kidney it has been shown that fluid movement may occur against a sodium concentration gradient across the proximal tubular waIl [4]. Therefore, it has been suggested that active transport of sodium provides the driving force for transepithelial reabsorption. To test whether the presence of sodium is critical for transepithelial net transport, isolated Necturus kidney was perfused in situ with various isotonic Na-free solutions. Stationary microperfusion experiments were carried out with the use of aliquots of the perfused solution, and the

252

253

Split-drops in Necturus perfused with Na-free media Table 1. Composition of perfusion fluids

Solution

KCI "X" Cl MgCI2 CaCI2 KH2PO4 K2HPO4 NaHCO3 mmoles/liter mmoles/liter mmoles/liter mmoles/ liter mmoles/liter mmoles/liter mmoles/liter NaCl 90 NaCI

Sodium bicarbonate Sodium phosphate Lithium

LiCI 95 KCI

95 RbCI 95

Rubidium

Choline

—

—

10

5

1

1.8

20

400

0.4

2

—

—

1

1.8

20

400

0.4

2

—

—

1

1.8

20

400

0.4

2

—

—

1

1.8

20

400

0.4

2

—

—

1

1.8

20

400

0.4

2

—

—

1

1.8

20

400

0.4

2

—

—

1

1.8

20

400

95

Potassium

Cesium

PVP4O Glucose g/ liter mg/liter

CsCl 95 Choline Cl 95

retraction of split-drops was usually observed until at least one-half of the fluid was reabsorbed. The results indicate

that fluid movement occurs whether or not sodium is present in peritubular and intratubular perfusate. The reabsorptive rate is lowered in experiments in which l0 M ouabain was added to the perfusate. This suggests that the integrity of the cell is required for the observed fluid reabsorption. Methods

Adult Necturi were kept unfed at 4° C and used at room temperature during the months of October 1971 through May 1972. They were anesthetized with a solution of 1.5% urethane, and the kidneys were perfused according to the

technique described by Tanner and Kinter [30]. The stability of the preparation was obtained by securing it with four pins. Neither decapitation and decerebration nor protease was necessary as in a previous electrophysiological study [31]. Perfusion rates of 0.11 and 0.21 mI/mm for the portal vein and the aorta, respectively, were achieved by means of a single Harvard pump. These rates are close to those generally maintained when working with Necturus [30, 32] and they seemed to be in the vicinity of the physiolo-

gical circulation rate, as observed optically by the washout of red blood cells during the early phases of perfusion.

Isotonic solutions of 200 mOsm/liter were prepared every day, each animal being perfused with a single solution,

in both the aorta and portal vein. The standard perfusion fluid was a Ringer's phosphate solution similar to that described by Giebisch [2] and used in a previous paper [311. However, the pH of this solution when polyvinylpyrrolidone (PVP) was present was only 6.8. Therefore, control experiments were also done with the use of Ringer's bicarbonate

potassium-phosphate solution allows complete removal of sodium during ionic substitutions. The substituted solutions were of similar composition to the Ringer's K-phosphate solution in all but the Na content, which was entirely replaced, as shown in Table 1. Sodium was substituted with

either lithium, K, rubidium, cesium or choline. Measurements of osmolality were made with an osmometer (Advanced Instruments). In some animals perfused either with Na-phosphate Ringer's oi choline solutions, 10 M ouabain was added to the medium. In every experiment, intraluminal injections (split-drops) were carried out using an aliquot of

the perfused solution. Micropuncture determinations were carried out from

45 minutes through 8 hours after the beginning of the perfusion. The Na concentration was measured in the caval outflow in seven animals. During the first 90 minutes, caval fluid contained measurable amounts of Na, though the Na

concentration was less than 20 mEq/liter (14.5 mEq/liter SEM, N= 11). This Na value probably represents

cellular efflux and some contamination with residual plasma. The heavy ligatures on the legs and tail made the perfusion space as small as possible, thus preventing further contamination. After 90 minutes no significant amount of Na was found in the effluent (concentration, 7.4 mEq/liter sEM; N= 8). The split-drops were usually introduced into superficial segments of early and late proximal tubules. The tubule was filled with light mineral oil through a glomerular pipette, and a second (tubular) pipette was used to inject the test solution into the lumen. Care was taken to push the droplet

distally to the puncture site, in order to avoid leaks or contamination or both. Frequent aspersions with oil or with the perfusing solution allowed the surface to stay

at pH 7.4. Since no important difference was observed in the respective t 4 values, no further attempt was made to

moist. The segments chosen for micropuncture were as straight as possible, but it was considered more important to obtain

correct the pH with other buffers, since the Ringer's

large split-drops of a total length of 8 to 12 diameters, so

254

Villey I Anagnostopoulos

that fluid reabsorption could be assessed with accuracy.

Some split-drops were introduced into a slightly convoluted though strictly horizontal segment, but observation was always continued through the disappearance of more

Table 2. Representative experiments demonstrating the variability of t4- from one animal to another

Date

Cation

than one-half of the split-drop. In some experiments the length of the split-drop was measured until three-fourths

"Fast"

"Slow"

animals

animals

Tubule t

or even seven-eighths of the initial volume was reabsorbed.

Slight errors in the measurement of tubular volume may have occurred since tubular diameter is not constant. In the experiments in which ouabain was added to the perfused

solutions, observation of the split-drop was discontinued after 90 minutes if no significant reabsorption had occured by that time. The use of large split-drops required some modifications in the technique. There is no a priori reason to assume that

reabsorption should result in a decrease in the size of

11-5-71

Lithium

1

2 3 1-27-72

11-17-71

11-27-71

the split-drop exclusively along its axis, which would yield

Lithium

Choline

1

20

2

23

3

22

Choline

determined with a micrometer disc. Care was taken to relax the pressure after each measurement. To avoid unnecessary repetition of short-time pressure variations,

1

70

2 3 4

45 63

Cesium

1

2 3 12-8-71

2-10-72

80

4

44 60 70 60

1

100

2 3

4

70 70 50 20 90 70

5

45

1

2 3 11-11-71

tf

miii

20 12 22

a regularly shrinking split-drop. Actually, a decrease in diameter did occur as well in a number of experiments. To make volume measurements comparable from one droplet to another, gentle pressure had to be applied through the glomerular pipette before every optical determination, until the distal meniscus started moving. Then the length was

Tubule

miii

25 22 15

Cesium

Sodium bicarbonate

the number of optical observations was deliberately limited and done at random six to eight times every hour, depending

1

2 3

on the rate of reabsorption. Although this procedure decreased the precision in the shape of the slope, it was more

accurate for the assessment of fluid reabsorption. No correction for the presence of meniscus was necessary with large split-drops. The variability of t - values in

Table 2. There may be some inborn differences between "fast" and "slow" animals. Factors such as age, sex, size

amphibians and the great length/diameter ratio made it

of tubules, site of observation, number of hours of observation after the beginning of the experiment and the duration

pointless. Therefore, the corrections recently recommended [33, 34] were not applied in this study. The use of exponen-

of fasting could explain, at least in part, the variability which is always greater in amphibians than in mammals.

tial decay could be a matter of controversy. The concept of exponential reabsorption has been proposed to quantify the relationship between rate of reabsorption and flow rate in free flow [35] or reabsorptive rate and surface area in shrinking drops [36]. Integration provides an exponential

Moreover, differences in the preparation are also possible. The time necessary for a bolus of lissamine green injected

law for fluid movement, and the reabsorptive t value becomes an accurate index for a first order phenomenon. In large split-drops, however, the length decrease is not

in the portal vein to appear in the kidneys indicated that one kidney, usually the left, was perfused better than the other. With the size of the animals ranging from 110 to 200 g, and a constant rate of perfusion, it is possible that tubular reabsorption changes as a function of perfusion rate per kidney weight, as in mammals.

so regular since retraction occurs in three dimensions.

In a first set of experiments (N= 108), the control re-

Hence, t 4 has to be considered to be an arbitrary but useful

absorptive t 4- values were 50.1 mm (Ringer's bicarbonate),

index if it is always measured. Accordingly, the results are presented in the conventional way.

and 54.6 mm (Ringer's phosphate), as compared to values of 42.3 mm (Li), 54.7 mm (K), 61.3 mm (Rb), 52.3 mm (Cs)

Results

In most micropuncture studies in Necturus, there is a great scatter in the data [3, 4, 32, 37]. Although uniform results can quite frequently be reproduced in a single animal, they appear to differ from one animal to another. Typical experiments with regard to this are shown in

and 51.2 mm (choline). The SEM and number of experiments are given in Table 3. In this series about 15 split-drops were

interrupted for technical reasons before the t 4- value was attained and, therefore, discarded. Fluid reabsorption in

these droplets was very slow, and after 20 minutes the degree of retraction was small enough to cast some doubt on the technique. Slight differences between the osmolality of Necturus plasma (range, 195 to 211 mOsm/liter; mean,

255

Split-drops in Necturus perfused with Na-free media Table 5. Summary of the complete t4 dataa

Table 3. Split-drop findings during cationic substitutions in the first set of experiments

Cation

N

Mean t4 SEM

Cation

mm

Sodium bicarbonate Sodium phosphate Lithium Potassium Rubidium Cesium Choline

50.1 4.8 54.6 4.7

Sodium bicarbonate Sodium phosphate Lithium Potassium Rubidium Cesium Choline

15

9

42.3 4.0 54.7 4.4

25

61.3±5.6

16 12 18

13

52.3 7.6 51.2± 7.1

Mean t4

Range of t4

mm

miii

SEM

Ani- Tumals bules

N

N

49.8

20 to 90

4.2

7

17

55.8

40 to 90

3.4

6

14

42.4 52.0

12 to 80 3Oto 90

3.9

10

26

3.8

6

56.5 50.2 54.8

26 to 120 15 to 100 15 to 130

5.0 4.8

10 10

17 22

6.3

7

22 23

a Table includes data from both first and second set of experi-

ments.

Table 4. Blind experiments

Date

Cation

t4

mm

Tubule 2-15-72

2-17-72

Cesium

Cesium

1

80

2

45

3

35

4

1

35 25 52 80 40 62

2

55

3

40 62 70 90

5 1

2 3

2-21-72

Sodium phosphate

4 5

2-19-72

3-30-72 4-1-72 4-1-72

Choline

Lithium Rubidium Rubidium

1

2

45

3

110

4 5

39 55 45 36 35 32

1 1 1

2 3-31-72

Rubidium

3 1

2 4-3-72

Potassium

1

2 3

4 4-4-72

Cesium

1

2 4-5-72

Sodium bicarbonate

1

2

90 35 35 33

30 50 60 45 40 45 50

204±2 SEM; N=9) and that of the perfusing solution

(range, 190 to 215 mOsm/liter; mean, 205

1.3 sEM;

N= 20) might have explained slow reabsorption in the first 20 minutes.

In a second series blind experiments were carried out in 11 additional animals in which a) the nature of the cations used was not known by the observer until the complete series was achieved, b) no measurement was initiated unless the technical conditions were excellent and c) the observation was continued up to 90 minutes even if no reabsorption appeared to occur in the first 20 minutes. With the exception of one droplet which was lost into the

distal tubule, the complete results of these 33 additional split-drops are shown in Table 4. It can be seen that there are no important differences in t 4 values between the first and the second sets of experiments. Table 5 shows average t 4 values of the pooled data from both sets of experiments (N= 141). The differences in t 4 values between any two cationic solutions are comparable to those found in the first set of experiments. The mean t 4 values obtained with control solutions are in good agreement with earlier observations on isolated perfused kidneys. The t4 values were 49.8 mm with the use of Ringer's bicarbonate and 55.8 3.4 mm with Ringer's phosphate, as compared to a value of 57 mm found in a previous study [32]. These results are similar to those obtained in intact summer Necturi (51 mm) [37], though t 4 values of 32 and 30 mm have also been reported in nonperfused animals [32, 37]. Despite the difference in pH of the two control solutions (7.4 for bicarbonate and 6.8 for phosphate), no significant

difference was found in the corresponding tf values (P>0.8). This observation suggests that pH variations, at least within this range, are less important than amphibian variability.

The mean reabsorptive t 4

values

for the substituted

solutions are 42 mm (Li), 52 mm (K), 56 mm (Rb), 50 mm (Cs) and 55 mm (choline). There is no significant difference between results of the Na-free experiments and the controls. Results of the experiments in which ouabain was added

to the perfused solutions are given in detail in Table 6. It clearly appears that transepithelial reabsorption is considerably slower in this series than in control ouabain-

256

Villey / Anagnostopoulos

Table 6. Effects of the use of 1O M ouabain on reabsorption t- values Date

t-

Tubule

mm

2

67 200 230

3

120

1 1

3-15-73 3-23-73

1

110

1

320

2 3

200 220

Choline and ouabain 3-12-73

1

2 3

3-14-73

3-16-73

I

125

240

1

No reabsorption No reabsorption 67 30 No reabsorption No reabsorption No reabsorption No reabsorption

1

2 3-20-73

1

2 3

3-21-73

a Mean

1

5EM

= 183

No reabsorption No reabsorption 40

2 3

2 3-19-73

integrity of the tissue is required for split-drop retraction, the latter perhaps reflecting effective tubular reabsorption. Nevertheless, the stop-flow techniques bear some short-

comings. It has been reported, for instance, that intratubular pressure in rats increases from 10 to 20 cm H2O

Sodium-phosphate and ouabain a 3-7-73 3-8-73

significant reabsorption, indicating that the functional

190

28.

free experiments. Ouabain-induced inhibition of proximal tubular reabsorption has already been reported in Necturus [38]. Table 6 shows that, despite variation from one experiment to another, transepithelial reabsorption is reduced whether the kidney is perfused with Na-phosphate Ringer's or with a choline solution. No average value is given for the latter, since no reabsorption could be detected within the first 90 minutes in several split-drops.

Discussion

To consider as fluid reabsorption the observed retraction

of oil split-drops in Na-free solutions, one must rule out any aitifact due to the technique. Since the technique used to study t with Na-Ringer's and with Na-free solutions was the same, the presence of an artifact is less likely. In addition, the series of blind experiments, in which the composition of the perfused fluid was unknown to the observer, makes this possibility even more remote. One may

argue, of course, that a systematic error might have been introduced and that the split-drop technique is less useful for this reason. The ouabain experiments, in which the same technique was applied, were not accompanied by

(free-flow) to 30 to 50 cm H20 (stop-flow) [39]. It is likely that similar pressure gradients occur in Necturus. However,

additional pressure was applied to the perfusate columns before each reading in the present experiments since, in several instances, fluid reabsorption resulted in a measurable

decrease in diameter and partial collapse of the tubule without concomitant change in the length of the split-drop.

Furthermore, on occasion a sudden reaspiration of the droplet after one hour of observation brought about a complete collapse of the tubule without any movement of the oil columns. Thus, we applied pressure to the perfusate columns during length measurements, in order to increase tubular diameter to a constant value; only then is length proportional to volume. Immediately after length determination the pressure was released until the next measurement. Whether the split-drop technique per se or the intermittent application of pressure just described, or both, may account partly for the observed reabsorption in Na-free experiments cannot be ascertained at present, though studies in mammals failed to demonstrate significant reabsorption after establishment of pressure gradients [40]. It is safer, however, not to rule out the possibility that small, although significant, differences in transport rate may have escaped detection in this study because of a) the wide scatter of the data and b) pressure gradients randomly exaggerated and! or permeability changes during the Na-free experiments. Even so, a sizable amount of reabsorption occurs in tests utilizing Na-free solutions and it appears to be linked to the metabolic activity of the cell, since fluid reabsorption was considerably reduced when i0 M ouabain was added to the perfusing solutions, though the same technique and similar pressure gradients were applied in these experiments. No oncotic pressure gradients were established across tubular epithelium since each split-drop was an aliquot of

the peritubular perfusate. Therefore, intratubular PVP concentration should have been equal to or greater than peritubular concentration at any given point in time. If the intraluminal concentration of sodium chloride is lowered by substitution of mannitol for NaCI, transepithelial net transport decreases proportionally [4]. In this

case passive efflux of NaCI from lumen to peritubular spaces decreases, whereas passive influx remains unchanged. This results in the net passive movement of NaCI inwards.

Since no osmotic gradient can be established across the proximal tubule under these conditions, the passive NaCI influx is isosmotic and compensates for the outwards fluid and electrolyte reabsorption which occurs despite an unfavorable transepithelial NaCI concentration gradient. The "zero net flux" concentration gradient for NaCI has been

257

Split-drops in Necturus perfused with Na-free media

estimated in several laboratories and in different species [5—7, 41]. This concentration gradient does not vary when peritubularNaCl concentration is modified [42]. Such experiments demonstrate the existence of fluid reabsorption against an electrochemical gradient and allow a quantitative estimate

of the driving force. They do not allow a firm conclusion about the nature of the driving force. Generally, they have been taken as significant evidence favoring primary Na reabsorption followed by water movement, rather than

by nonspecific poisoning of the cells. In either case the reabsorption rate observed in the absence of ouabain does reflect biological transport. Thus, we conclude that transepithelial electrolyte transport in Necturus lacks specificity,

though it is not known whether the processes for fluid reabsorption are identical in Na-Ringer's and Na-free solutions. If so, a nonspecific coupled water-electrolyte transport, metabolically linked, might be considered as an alternative working hypothesis.

primary water transport. However, the possibility of a coupled transport of NaCl and water, or, more generally, solute-water coupled transport, has received less attention. Ionic substitutions have been used extensively to study electrical properties of renal tubules. The effects of ionic substances on fluid reabsorption were studied to a smaller extent. Replacement of chloride by cyclosulfaminate in rats increases reabsorptive t4 value from 40 to 103 sec [43], i.e., a 2.7-fold increase. Substituting choline for sodium intraluminally and peritubularly in rat kidney slices decreases the reabsorptive rate to the same extent, since the reported t-- values were 49 and 22 sec, respectively (i.e., a 2.2-fold increase in t value during choline substitution) [41]. The

observation that removal of either Na or Cl is equally effective in curtailing fluid reabsorption and the persistence

of reabsorption in Na-free media in rats are hardly con-

Acknowledgments

This work was supported by a grant from INSERM. Reprint requests to Dr. T. Anagnostopoulos, Hôpital des Enfants-Malades (INSERM U. 30), Tour Technique — 6e étage, 149, rue de Sèvres, Paris 15e, France.

1.

1958 2. GIEBISCH G: Measurements of electrical potential differences

on single nephrons of the perfused Necturus kidney. J Gen Physiol 44:659—678, 1961 3. OKEN DE, WHITTEMBURY G, WINDHAGER EE, SCHATZMANN

HJ, SOLOMON AK: Active sodium transport by the proximal tubule of Necturus

sistent with the scheme of a specific sodium pump mediating

reabsorption. In regard to other tissues, Li net transport has also been reported in frog skin [44] and toad urinary

possible changes in reabsorptive rate but to find out if transepithelial transport ceases in the absence of sodium. The present data indicate that reabsorption is not suppressed by removal of sodium and, also, that the rate of choline reabsorption is lowered by addition of lO M ouabain. The detailed mechanism of these observations cannot be ascertained with accuracy at present. Briefly, the characteristics of Na-free fluid transport are as follows: a) it cannot be accounted for entirely by physical forces, i.e., hydrostatic

or oncotic pressure gradients, or both, for the reasons already discussed; b) no transepithelial chemical gradient was established by the experimental procedure we used, and no electrical gradient was detected in experiments in

which Li and choline were used (T. Anagnostopoulos, D. Villey and M. Claret, unpublished data); and c) when

10 M ouabain was added to the perfusing solutions, splitdrop reabsorptive t values were consistently increased in experiments in which both sodium and choline were used. Since the tissue was exposed to high concentrations of ouabain for several hours, it cannot be assessed from these experiments whether ouabain specifically inhibits fluid reabsorption as reported by others [38] or whether it acts

kidney.

J C/in Invest 38:1029, 1959

4. WINDHAGER EE, WHITTEMBURY G, OKEN DE, SCHATZMANN

HJ, SOLOMON AK: Single proximal tubules of the Necturus

III. Dependence of H20 movement on NaCI concentration. Am J Physiol 197:313—318, 1959 KOKKO JP, BURG MB, ORLOFF J: Characteristics of NaCI kidney:

bladder [45].

Our observations indicated that replacement of sodium with various cations does not impede fluid reabsorption in Necturus. There is no great change in reabsorptive rate, though the latter must be taken only as a rough estimate. Indeed, the main purpose of this study was not to detect

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