- Email: [email protected]
Effect of expansion of extracellular fluid volume on proximal sodium reabsorption in hyponatremic dogs
Effect of Expansion of Extracellular Fluid Volume on Proximal Sodium Reabsorption in Hyponatremic Dogs By BERNARDB. DAVIS, FRANKLYN G. KNOX, FRED STUART S. HOWARDS
The effects of infusions of equivalent vohtmes of saline solutions on proximal tubule sodium reabsorption and sodium excretion were studied in normonatremic and hyponatremic dogs. Similar expansion of the extracelhthu fluid volumes resulted in 48.1 per cent t 3.63 per cent and 19.4 per cent 2 7.45 per cent decreases in proximal tubule reabsorption in the normonatremic and hyponatremic dogs respectively (p < 0.005). Sodium excretions increased from 7.3 + SE 1.78 rEq./min. to 289 h SE 45.9
S. WRIGHT AND
cEq./min. in normal dogs (p NA 144 mEq./L.) and from 3.1 _+0.87 &Eq./min. to 63.2 f 23.0 pEq./min. in hyponatremic dogs (p Na 126 mEq./L.) following infusion of saline sohrtfons. Expansion of the extracefhdar fluid volume of the hyponatremic dogs resulted in a significantly smaller decrease in sodium reabsorption by the proximal tubule and a signifwantly smaller increase in sodium excretion than in normonatremic dogs. (Metabolism 19: No. 4, April, 291-300, 1970)
THE INTRAVENOUS INFUSION of saline solution to dogs results in a l_ marked increase in the rate of sodium excretion. Blythe and Welt’ have shown that the increase in sodium excretion accompanying infusion of saline solution was reversed by water loading. Since the filtered load of sodium was not decreased, the change in sodium excretion was attributed to an increase in tubule sodium reabsorption as a consequence of the hyponatremia induced by water loading. Schrier et al .2 have demonstrated that expansion of the plasma volume by infusion of dextran in saline solution increased sodium excretion whereas equivalent expansion of the plasma volume by infusion of dextran in glucose solution did not significantly increase sodium excretion. One interpretation offered for these results by Schrier et al. was that, although expansion of the intravascular volume was a stimulus to increase sodium excretion, significant natriuresis was prevented by reduction of the sodium concentration in the extracellular fluid. More recently Schrier and co-workers3 have presented further evidence to support the hypothesis that hyponatremia has an antinatriuretic From the Laboratory of Kidney and Electrolyte Metabolism, National lnstitufes of Health, Bethesda, Md., and the Department of Pittsburgh, Pittsburgh, Pa. Received Supported
for publication
October
in part by USPHS
National Heart Institute, of Medicine, University
16, 1%9.
Grant AM 11911-02
B. DAVIS, M.D.: Assistant
from
the National
Institutes of Health.
of Medicine, University of Pittsburgh, Pittsburgh, Pa. FRANKLYN G. KNOX, M.D., PH.D.: Assistant Professor of Physiology, University of Missouri School of Medicine, 3. Louis, MO. FRED WRIGHT, M.D.: Instructor in Physiology, Yale University School of Medicine, New Haven, Conn. STUART HOWARDS, M.D.: Resident in Wrology, Peter Bent Brigham Hospital, Boston, Mass. BERNARD
METABOLISM, VOL. 19, No. 4 (APRIL),
Professor
1970
291
292
DAVIS ET AL.
effect which counterbalances the natriuretic property associated with the infusion of saline solutions. One concomitant of the intravenous infusion of saline or dextran solutions is a decrease in the fractional reabsorption of sodium by the proximal tubule.4vS The following experiments were performed to compare the effects of similar expansion of the extracellular fluid volume on fractional reabsorption of sodium by the proximal tubule in normal and hyponatremic dogs. Infusion of equivalent volumes of saline solutions which were made isotonic to the dog resulted in both a significantly smaller decrease in fractional sodium reabsorption by the proximal tubule and significantly less increase in sodium excretion in hyponatremic than in normonatremic dogs. Both of these parameters were returned to control levels when the plasma sodium of hyponatremic dogs was restored to normal levels. The data identify the proximal tubule as a site of altered sodium reabsorption in response to isotonic extracellular fluid volume expansion in hyponatremic dogs. MATERIALS
AND METHODS
Hyponatremia was induced and maintained in dogs by a regimen consisting of administration of 25 mg. of ethacrynic acid for two days, daily administration of water (by gastric tube) with injection of 5 U of vasopressin in oil to maintain initial weight, and feeding a low sodium diet for seven days. Seven normonatremic and 19 hyponatremic dogs were deprived of food and water for 18 hours prior to the experiments. Anesthesia was induced and maintained with pentobarbital. The micropuncture technique, in which samples were obtained from proximal tubules during hydropenia and from the same segments after a change in experimental conditions, has been previously described.6 Care was taken during the collection of samples so that a previously injected drop of polymer oil (Kel-F) remained stationary distal to the point of collection in the tubule. Four groups of experiments were done.
Normal Dogs with Zsotonic Saline During hydropenia, samples of proximal tubule fluid were obtained while three 15minute collections of urine were made. After this 45minute control period, an intravenous infusion of 0.85 per cent saline was begun at a rate of 1.0 cc./Kg./min. After 20 minutes the infusion rate was reduced to 0.5 cc./Kg./min. for the remainder of the experiment. Thirty-eight minutes after the reduction of the infusion rate, three additional 15-minute urine collections were obtained. During this 45-minute period, tubule fluid was obtained from the same proximal tubule sites sampled during hydropenia. During these collections the saline infusion was continued at a rate of 0.50 cc./Kg./min.
Hyponatremic
Dogs with Hypotonic Saline
Proximal tubule fluid samples and urine collections for clearances were obtained during hydropenia. The plasma sodium concentration was determined and a saline solution was prepared with a sodium concentration equal to that of the plasma. This solution was given intravenously at the same rates as the physiologic saline had been administered to the normal dogs. Fluid samples were obtained from the same tubule sites and three urine collections were obtained for clearance determinations. A representative experiment is shown in Table 1.
Hyponatremic
Dogs with Hypertonic Saline
Tubule fluid samples and urine for clearance determinations were obtained during hydropenia. Ten per cent sodium chloride solution was then infused for 20 minutes at a rate of 0.1 cc./Kg./min., an amount calculated to expand the extracellular fluid volume 20 ml./Kg. as
of NaCl of NaCl
48.4
65.8
126.1
3.18
3.69
4.36 131
128
124
solution, solution,
710
infusion infusion
1.1
Intravenous Intravenous
.22
730
1.4
.22
720
LJ0.m
1.2
~
Na Excr. (pEq./min.)
.21
Urine Flow (ml./min.) ___
60
60
68
P Inulin (w4 70)
Hypotonic
279
276
278
54
47
50
Na Concentration Na Concentration
277
277
278
P<,S”,
1 .-Protocol:
97.0
98.0
91.4
130 mEq./L. 130 mEq./L.
79.8
88.0
71.4
C Inulin (ml./min.) ____ -
85 109
88 89 86
83
93 110
lyI$$ 0
1.42 1.82
1.42 1.48 1.43
1.26
1.37 1.64
TF/P lnulin ___
to Hyponatremic
Dog*
65 75
70 61
67 66
1.35 1.53
1.45 1.30
1.37 1.37
at rate of 14.6 ml./min. at rate of 7.3 ml./min. 62 1.25 90 1.81
Saline Infusion
__
26 35
31 23
27 27
20 45
30 45
30 32 30
21
27 39
‘XI Fract. Reab. ~~ ~__
-.
-13 -22
-3 -23
+29 -10
-26 +15
% Change Fract. Reab. ..~ _
7b 8b
5b 6b
3b 4b
lb 2b
7a 8a
4a 5a 6a
3a
la 2a
T.F. St3IlWlC Number ~~ -
-7% Mean ~~___ _ .._.___~ * Initial dog weight: 14.8 Kg. Day of experiment: 14.6 Kg. P,, 130 mEq./L. U 08111=urine osmolality mOsm/Gm. H,O; POR,,,=plasma osmolality mOsm/Gm. H,O; P inulin=plasma inulin concentration; C inulin=inulin clearance; TF inulin=concentration of inulin in tubule fluid: % fract. reab.= per cent of glomerular filtrate reabsorbed to point of micropuncture.
66-104 107 110 111 113 116 119 121 124 126 128 132 134 141 149
2 6 7 12 15 16 22 27 30 31 37 45 46-66
(min.)
yP$
Table
294
DAVIS
ET AL.
was done in Groups I and II. Isotonic saline was then infused at a rate of 0.5 ml./Kg. for the remainder of the experiment. Recollection of tubule fluid samples and urine for clearances were obtained as in the first two groups. In the fourth group of six additional dogs, the volume of extracellular fluid was estimated from the volume of distribution of 500 mg. of inulin given by intravenous injection before and after the induction of hyponatremia. Correction was made for loss of inulin in the urine. In all animals venous blood samples were collected at the midpoint of each urine collection period from an indwelling femoral vein catheter; urine was collected through ureteral catheters. The concentration of inulin in tubule fluid (TF In) and plasma (P In) samples was determined. The tubule fluid to plasma inulin concentration ratios (TF/P In) were calculated by interpolating the observed plasma inulin concentrations to the time of collection of the tubule fluid. Fractional reabsorption of glomerular filtrate to the point of micropuncture was calculated from the formula l-(P/TF In). For statistical evaluation, the mean change in fractional reabsorption, obtained from three to nine tubules in each dog, was considered as a single observation. Inulin was measured in tubule fluid by the microfluorometric method of Vurek and Pegram,? and in plasma and urine by the anthrone method of Fiihr et al.8 Sodium in plasma and urine was determined by flame photometry using a lithium internal standard. Plasma and urine osmolalities were measured with a Bowman osmometer. RESULTS
In the 7 normonatremic dogs, average sodium concentration 144 mEq./L., 43 samples of fluid from proximal tubules were obtained during hydropenia. After infusion of saline, fluid was collected from the same segments. The concentration ratios of inulin in tubule fluid to that in plasma (TF/P) of these 43 paired samples are plotted in Fig. 1. The mean ratio of the (TF/P) during saline loading to that in hydropenia was 0.825 + 0.065. The mean decrease in fractional sodium reabsorption calculated from these (TF/P) inulin ratios was 48.1 per cent -c 3.63 per cent. Sodium excretion rose from a mean of 7.3 f SE 1.78 pEq./min. during the control periods to a mean of 289 + SE 45.9 pEq./ 2.5 -
NORMAL f
ISOTONIC
2.0-
DOGS SALINE
HYOROPENIA
Fig. I.-Proximal tubule sodium reabsorption. Points represent paired collections of proximal tubule fluid with value of (TF/P) inulin during hydropenia plotted against value after infusion. Solid line indicates no change; broken line indicates mean Effect of infusion of isotonic saline change from control to experimental collections. to normal dogs.
EXTRACELLULAR
FLUID
VOLUME
295
EXPANSION
.0HYPONATREMIC HYPOTONIC
DOGS SALINE
A = MEAN
1.0
I. 5
2.0 1111111 Ihl
2.5
HYDROPENIA
Fig. 2.-Proximal tubule sodium reabsorption. Points represent paired collections of proximal tubule fluid with value of (TF/P) inulin during hydropenia plotted against value after infusion. Solid line indicates no change; broken line indicates mean change from control to experimental collections. Effect of infusion of hypotonic saline to hyponatremic dogs.
min. during experimental periods. The urine volume increased from 0.20 r+ SE 0.019 ml./min. to 2.6 rf: SE 0.15 ml./min. after the saline infusion. The mean inulin clearance was 58.2 & SE 5.0 ml./min. during control and 68.4 Z!XSE 5.8 ml./min. in the experimental periods. The mean weight of the dogs before induction of hyponatremia was 14.43 +SE 0.53 Kg., the mean weight on the day of the experiment was 14.40 + SE 0.52. The change in weight of -0.03 was not significant. In the seven hyponatremic dogs, average sodium concentration 126 mEq./L., 38 pairs of tubule fluid samples were obtained. These data are plotted in Fig. 2. The mean ratio of the (TF/P) inulin after the saline to that during hydropenia was 0.915 + SE 0.022. This represents a depression of fractional sodium reabsorption by the proximal tubule of 19.4 per cent rt SE 7.45 per cent, a significantly smaller decrease in fractional reabsorption than was measured in the normal dogs (p < 0.005; see Table 2). Sodium excretion increased from 3.1 k SE 0.87 pEq./min. during hydropenia to 63.2 t SE 23.0 pEq./min. after saline. This rise is significantly less than that observed in the normonatremic dogs (p < 0.0 1) . Urine flow increased from 0.30 2 SE 0.047 ml. to 3.53 -t SE 0.88 ml./min. This increase is not significantly different from the increase noted in the normal dogs. Inulin clearance increased from 66.7 * 5.77 ml./min. to 71 f 5.68 ml./ min. As shown in Table 3, there was no significant change in the plasma osmolality following the infusions; however, urine osmolality changed from hypertonic to hypotonic in each experiment. The data from 42 pairs of samples obtained from six hyponatremic dogs given hypertonic saline are plotted in Fig. 3. The mean ratio of the TF/P In after
296
DAVIS ET AL.
Table 2.-Mean
per Cent of Proximal Na Reabsorption Following Saline Infusion
Normal Dogs Isotonic Saline n tubules
(5)
-49 -59 -59 -37 -45 -52 -36
(7) (8) (6) (6) (4) (7)
Hyponatremic Dogs Hypertonic Saline n tubules
-10 -13 -31 -59 -15 -1 -7
-64 -22 -51 -55 -35 -64
(5) (5) (3) (6) (7) (4) (8)
- 19.4 k 7.47 7
-48.1 SE + 3.63 7 n dogs
I
Hyponatremic Dogs Hypotonic Saline n tubules
(7) (6) (7) (9) (7) (6)
-48.5 + 5.57 6
p < 0.005
I
I
I P < 0.02 Table 3.-Effect of Infusion of Hypotouic or Hypertonic Saiine to Hyponatremic Dogs on Plasma and Urine Osmoiaiities, and Plasma Sodium Concentration
H
Dog
P mOsm/Kg.
A. Hypotonic
Sa;Ii
S
H
zfy?Ln . .
S
H
U mOsm/Kg.
S
1 2 3 4 5 6 7
278 273 275 257 261 255 277
277 270 274 260 261 250 278
132 116 130 123 125 124 131
134 123 133 128 126 124 135
700 633 347 1000 1180 490 720
61 115 229 66 94 213 130
Mean
268
267
126
129
724
130
Doe
H
B. Hypertonic
1 2 3 4 5 6 Mean
P mOsm/Kg.
Saline Infusion PN= mEq./L.
U mOsm/Kg.
S
H
S
H
270 259 275 274 263 270
287 295 322 285 286 300
132 129 134 128 124 133
142 144 157 139 142 151
359 920 131 900 1098 294
135 96 171 750 270 66
269
296
130
146
617
248
H =Hydropenia;
S
S = Saline; P = Plasma; U = Urine.
saline infusion to that during hydropenia was 0.826 k SE 0.018. The mean decrease in fractional sodium reabsorption calculated from these figures was 48.5 I+ SE 3.6 per cent. This is not significantly different from the depression following saline infusion in normal dogs. It is, however, a significantly greater depression than that noted in hyponatremic dogs (p < 0.02; Table 2). Sodium excretion rose from 61.5 k SE 4.33 pEq./min. to 204 k 140. Urine flow rate
EXTRACELLULAR
-
FLUID VOLUME
297
EXPANSION
2.5 -
u U-J
HYPONATREMIC HYPERTONIC
DOGS SALINE
A= MEAN
,NUL,N HYDROPENIA
Fig. 3.-Proximal tubule sodium reabsorption. Points represent paired collections of proximal tubule fluid with value of (TF/P) inulin during hydropenia plotted against value after infusion. Solid line indicates no change; broken line indicates mean change from control to experimental collections. Effect of infusion of hypertonic saline to hyponatremia dogs.
from 0.66 SE + 0.038 cc./min. to 4.58 rt 1.82, neither increase being significantly different from the response to saline infusion in normal dogs. The mean inulin clearance was 50.5 cc./min. during hydropenia and this rose to 54.0 cc./min. after the hypertonic saline. The hypertonic saline caused an increase in the mean plasma osmolality from 269 to 296 mOSm/Gm. Hz0 and the mean plasma sodium from 130 to 146 mEq./L. (Table 3). The results from the six additional dogs in which plasma sodium concentration, inulin space measurements, and weights were measured before and after production of chronic hyponatremia are shown in Table 4. The change in plasma sodium concentration - 14.5 -+ SE 3.3 mEq./L. was significant (p < .Ol ) . The increased
Dog
P!yna
Table
4.-E&&
Sodiim
(mEq./k)
of Production
256
148
- 108
14.5
15.0
-3 +68 +103 -112 -32
12.0 13.0 11.5 15.0 12.0
12.0 13.3 11.7 15.0 11.9
13.0 0.6
13.1 0.6
C
1
142
135
-7
2 3 4 5 6
146 143 150 147 145
120 132 139 124 136
-26 -11 -11 -23 -9
287 134 193 218 222
284 202 296 106 190
145.5 1.1
131.0 3.0
- 14.5 3.3 < .Ol
218 22
204 30
Mean tlSE
of Chronic
Inulin Spage (ml./Kgh)
__C = Control;
H = Hyponatremia.
-14 36 N.S.
Hyponatremia C
Weigz
(Kg.)
A
+o.s 0.0 +0.3 +0.2 0.0 -0.1 +0.15 .09 N.S.
298
DAVIS ET AL.
changes in inulin space, - 14 SE _+ 36 ml/Kg. .09 Kg. were not significant.
and dog weight, f0.15
I
SE
DISCUSSION
Intravenous saline was administered to both normal and chronically hyponatremic dogs. Isotonic (.85%) saline was infused into the normals. The sodium concentration of the fluid given to the hyponatremic animals was adjusted so that the solution was isotonic to the recipient. Equivalent volumes were administered to both groups but the saline caused less depression of fractional sodium reabsorption by the proximal tubule in hyponatremic than in normal dogs. The saline also resulted in a lesser increase in sodium excretion in the hyponatremic group. These findings are in accord with those of Blythe and Welt1 and Schrier et a1.2 and localize the antinatriuretic effect of hyponatremia at least in part to the proximal tubule. The similarity in body weight before and after induction of hyponatremia indicates that total body water was unchanged by the procedure. That extracellular fluid volume remained constant is supported by the finding that there was no measurable change in the inulin space with the production of hyponatremia. The variability in these measurements is such, however, that a small change in extracellular fluid volume might have gone undetected. Fractional sodium reabsorption in the proximal tubule was similar in both normal and hyponatremic dogs during hydropenia. Therefore, starting from comparable baselines expansion of the extracellular fluid volume resulted in a significantly smaller decrease in sodium reabsorption by the proximal tubule in hyponatremic dogs than in those with a normal serum sodium. When extracellular fluid volume was expanded concomitant with restoration of the serum sodium to normal, hyponatremic dogs infused with hypertonic saline, this difference was abolished. The interpretation of these data is that hyponatremia acted to decrease the response of the proximal tubule to isotonic expansion of the extracellular fluid volume. The result of this effect of hyponatremia was that the proximal tubules of the hyponatremic dogs reabsorbed a greater fraction of the glomerular filtrate after saline infusion than did the proximal tubules of the normal animals. We have previously reported experiments in which 5 per cent albumin in saline solution was first equilibrated with the blood volume of dogs resulting in dilution of the blood without changes in vascular volume and subsequently the blood volume was expanded without changes in the composition of the blood.g It was concluded that both dilution of the blood and expansion of the blood volume are independent and important factors in the decrease in sodium reabsorption by the proximal tubule following infusion of saline solution. In the present series of experiments both isotonic solutions used would be expected to result in equivalent dilution and expansion of the blood volumes. The significantly smaller decrease in sodium reabsorption by the proximal tubules of hyponatremic dogs could be related therefore to a blunted response to either dilution or expansion. The mechanism of the decreased sodium reabsorption by the proximal tubule that is induced by intravenous saline is controversial. Two major hypothesis have been advanced. One holds that the depression in fractional sodium reabsorption
299
EXTRACELLULAR FLUID VOLUME EXPANSION
I
40 FRACTIONAL S00lUM REABSORPTION BY PROXIMAL TUBULE K
30
-
20
-
10 -
+h *
Ii
H
DE
DE
0 HYPONATREMIC
NORMONATREMIC
H
D
DE
NORMONATREMIC
DOGS
Fig. 4.-Fractional reabsorption +-1 SE by proximal tubule of normonatremic and hyponatremic dogs during continued hydropenia (H) and following dilution alone (D) or dilution and expansion (DE) of blood volume.
is mediated by an altered activity of an unknown hormone. The other suggests that the effect is mediated by a change in the physico-chemical properties of the blood perfusing the proximal tubules. The increase in sodium excretion of recipient dogs in cross circulation experiments in which the donor dog was saline loaded supports the hypothesis that infusion of saline results in a change in a humoral factor.lO-‘” Rector et al .13 have presented evidence for a humoral agent which decreases sodium reabsorption by the proximal tubule of the rat following extracellular fluid volume expansion. However, since Schrier et al.l* have been unable to confirm these results, the existence of such a hormone remains speculative. It would be expected that changes in physical factors, such as the colloid osmotic pressure in peritubular capillaries, which are thought to be responsible for the decreased proximal reabsorption during dilution would have been similar in hyponatremic and normal dogs. If that is true, the hyponatremic dogs might have responded to dilution but not expansion of the extracellular fluid volume. Support for this suggestion is found in Fig. 4 where the effects of dilution and expansion of the blood volume from the dogs previously reported9 is compared to the data obtained in these experiments. Dilution of the blood volume without expansion resulted in a depression of fractional sodium reabsorption similar to that measured in the hyponatremic dogs that received an isotonic infusion. The results of both expansion and dilution were similar to the response of the normal dogs in these experiments. ACKNOWLEDGMENT We are grateful for the invaluable technical assistance of Nordica Green and Marilyn J. Walter.
REFERENCES 1. Blythe, W. B., and Welt, L. G.: Plasma sodium concentrations and urinary sodium excretion. Trans. Ass. Amer. Physicians 78: 90-96, 1965. 2. Schrier, R. W., McDonald, K. M., Marshall, R. A., and Lauler, D. P.: Absence of natriuretic response to acute hypotonic intravascular volume expansion. Clin. Sci. 34: 57-72, 1968. 3. -, Fein, R. L., McNeil, J. S., and Cirksena, W. J.: Influence of interstitial fluid
volume expansion and plasma sodium concentration on the natriuretic response to volume expansion in dogs. Clin. Sci. 36: 371-385, 1969. 4. Dirks, J. H., Cirksena, W. J., and Berliner, R. W.: The effect of saline infusion on sodium reabsorption by the proximal tubule of the dog. J. Clin. Invest. 44: 1160-1170, 1965. 5. Howards, S. S., Davis, B. B., Knox, F. G., Wright, F. S., and Berliner, R. W.: De-
300 pression of fractional sodium reabsorption by the proximal tubule of the dog without sodium diuresis. I. Clin. Invest. 47:15161572, 1968. 6. Davis, B. B., Knox, F. G., and Berliner, R. W.: Effect of vasopressin on proximal tubule sodium reabsorption in the dog. Amer. J. Physiol. 212: 1361-1364, 1967. 7. Vurek, G., and Pegram, S.: Fluorometric method for the determination of nanogram quantities of inulin. Anal. Biochem. 16: 409-419, 1966. 8. Fiihr, J., Kaczmarcyzk, J., and Kruttgen, D. : Eine Einfache colorimetrische Methode zur Inulinbestimmung fur Nierenclearance-Untersuchungen bei Stoffwechselgesunden und Diabetickern. Clin. Wochschr. 33~729-730, 1955. 9. Knox, F. G., Howards, S. S., Wright, F. S., Davis, B. B., and Berliner, R. W.: Effect of dilution and expansion of blood volume on proximal sodium reabsorption. Amer. J. Physiol. 215:1041-1048, 1968. 10. Johnston, C. I., and Davis, J. 0.: Evidence from cross circulation studies for a humoral mechanism in the natriuresis of
DAVIS
ET AL.
saline loading. Proc. Sot. Exp. Biol. Med. 121: 1058-1063, 1966. 11. DeWardener, H. E., Mills, I. H., Clapham, W. F., and Hayter, C. J.: Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin. Sci. 21:249258, 1961. 12. Lichardus, B., and Pearce, J. W.: Evidence for a humoral natriuretic factor released by blood volume expansion. Nature 209:407-409, 1966. 13. Rector, F. C., Martinez-Maldonado, M., Kurtzman, N. A., Sellman, J. C., Oerther, F., and Seldin, D. W.: Demonstration of a hormonal inhibitor of proximal tubular reabsorption during expansion of extracellular volume with isotonic saline. J. Clin. Invest. 47:761-773, 1968. 14. Schrier, R. W., Verroust, P. J., DeWardener, H. E., Holzgreve, H., Brenner, B. M., Wright, F. S., Bennett, C. M., Keimowitz, R. I., and Berliner, R. W.: Failure to demonstrate a humoral inhibitor of proximal sodium reabsorption. J. Clin. Invest. 48: 1107, 1969.
The effects of infusions of equivalent vohtmes of saline solutions on proximal tubule sodium reabsorption and sodium excretion were studied in normonatremic and hyponatremic dogs. Similar expansion of the extracelhthu fluid volumes resulted in 48.1 per cent t 3.63 per cent and 19.4 per cent 2 7.45 per cent decreases in proximal tubule reabsorption in the normonatremic and hyponatremic dogs respectively (p < 0.005). Sodium excretions increased from 7.3 + SE 1.78 rEq./min. to 289 h SE 45.9
S. WRIGHT AND
cEq./min. in normal dogs (p NA 144 mEq./L.) and from 3.1 _+0.87 &Eq./min. to 63.2 f 23.0 pEq./min. in hyponatremic dogs (p Na 126 mEq./L.) following infusion of saline sohrtfons. Expansion of the extracefhdar fluid volume of the hyponatremic dogs resulted in a significantly smaller decrease in sodium reabsorption by the proximal tubule and a signifwantly smaller increase in sodium excretion than in normonatremic dogs. (Metabolism 19: No. 4, April, 291-300, 1970)
THE INTRAVENOUS INFUSION of saline solution to dogs results in a l_ marked increase in the rate of sodium excretion. Blythe and Welt’ have shown that the increase in sodium excretion accompanying infusion of saline solution was reversed by water loading. Since the filtered load of sodium was not decreased, the change in sodium excretion was attributed to an increase in tubule sodium reabsorption as a consequence of the hyponatremia induced by water loading. Schrier et al .2 have demonstrated that expansion of the plasma volume by infusion of dextran in saline solution increased sodium excretion whereas equivalent expansion of the plasma volume by infusion of dextran in glucose solution did not significantly increase sodium excretion. One interpretation offered for these results by Schrier et al. was that, although expansion of the intravascular volume was a stimulus to increase sodium excretion, significant natriuresis was prevented by reduction of the sodium concentration in the extracellular fluid. More recently Schrier and co-workers3 have presented further evidence to support the hypothesis that hyponatremia has an antinatriuretic From the Laboratory of Kidney and Electrolyte Metabolism, National lnstitufes of Health, Bethesda, Md., and the Department of Pittsburgh, Pittsburgh, Pa. Received Supported
for publication
October
in part by USPHS
National Heart Institute, of Medicine, University
16, 1%9.
Grant AM 11911-02
B. DAVIS, M.D.: Assistant
from
the National
Institutes of Health.
of Medicine, University of Pittsburgh, Pittsburgh, Pa. FRANKLYN G. KNOX, M.D., PH.D.: Assistant Professor of Physiology, University of Missouri School of Medicine, 3. Louis, MO. FRED WRIGHT, M.D.: Instructor in Physiology, Yale University School of Medicine, New Haven, Conn. STUART HOWARDS, M.D.: Resident in Wrology, Peter Bent Brigham Hospital, Boston, Mass. BERNARD
METABOLISM, VOL. 19, No. 4 (APRIL),
Professor
1970
291
292
DAVIS ET AL.
effect which counterbalances the natriuretic property associated with the infusion of saline solutions. One concomitant of the intravenous infusion of saline or dextran solutions is a decrease in the fractional reabsorption of sodium by the proximal tubule.4vS The following experiments were performed to compare the effects of similar expansion of the extracellular fluid volume on fractional reabsorption of sodium by the proximal tubule in normal and hyponatremic dogs. Infusion of equivalent volumes of saline solutions which were made isotonic to the dog resulted in both a significantly smaller decrease in fractional sodium reabsorption by the proximal tubule and significantly less increase in sodium excretion in hyponatremic than in normonatremic dogs. Both of these parameters were returned to control levels when the plasma sodium of hyponatremic dogs was restored to normal levels. The data identify the proximal tubule as a site of altered sodium reabsorption in response to isotonic extracellular fluid volume expansion in hyponatremic dogs. MATERIALS
AND METHODS
Hyponatremia was induced and maintained in dogs by a regimen consisting of administration of 25 mg. of ethacrynic acid for two days, daily administration of water (by gastric tube) with injection of 5 U of vasopressin in oil to maintain initial weight, and feeding a low sodium diet for seven days. Seven normonatremic and 19 hyponatremic dogs were deprived of food and water for 18 hours prior to the experiments. Anesthesia was induced and maintained with pentobarbital. The micropuncture technique, in which samples were obtained from proximal tubules during hydropenia and from the same segments after a change in experimental conditions, has been previously described.6 Care was taken during the collection of samples so that a previously injected drop of polymer oil (Kel-F) remained stationary distal to the point of collection in the tubule. Four groups of experiments were done.
Normal Dogs with Zsotonic Saline During hydropenia, samples of proximal tubule fluid were obtained while three 15minute collections of urine were made. After this 45minute control period, an intravenous infusion of 0.85 per cent saline was begun at a rate of 1.0 cc./Kg./min. After 20 minutes the infusion rate was reduced to 0.5 cc./Kg./min. for the remainder of the experiment. Thirty-eight minutes after the reduction of the infusion rate, three additional 15-minute urine collections were obtained. During this 45-minute period, tubule fluid was obtained from the same proximal tubule sites sampled during hydropenia. During these collections the saline infusion was continued at a rate of 0.50 cc./Kg./min.
Hyponatremic
Dogs with Hypotonic Saline
Proximal tubule fluid samples and urine collections for clearances were obtained during hydropenia. The plasma sodium concentration was determined and a saline solution was prepared with a sodium concentration equal to that of the plasma. This solution was given intravenously at the same rates as the physiologic saline had been administered to the normal dogs. Fluid samples were obtained from the same tubule sites and three urine collections were obtained for clearance determinations. A representative experiment is shown in Table 1.
Hyponatremic
Dogs with Hypertonic Saline
Tubule fluid samples and urine for clearance determinations were obtained during hydropenia. Ten per cent sodium chloride solution was then infused for 20 minutes at a rate of 0.1 cc./Kg./min., an amount calculated to expand the extracellular fluid volume 20 ml./Kg. as
of NaCl of NaCl
48.4
65.8
126.1
3.18
3.69
4.36 131
128
124
solution, solution,
710
infusion infusion
1.1
Intravenous Intravenous
.22
730
1.4
.22
720
LJ0.m
1.2
~
Na Excr. (pEq./min.)
.21
Urine Flow (ml./min.) ___
60
60
68
P Inulin (w4 70)
Hypotonic
279
276
278
54
47
50
Na Concentration Na Concentration
277
277
278
P<,S”,
1 .-Protocol:
97.0
98.0
91.4
130 mEq./L. 130 mEq./L.
79.8
88.0
71.4
C Inulin (ml./min.) ____ -
85 109
88 89 86
83
93 110
lyI$$ 0
1.42 1.82
1.42 1.48 1.43
1.26
1.37 1.64
TF/P lnulin ___
to Hyponatremic
Dog*
65 75
70 61
67 66
1.35 1.53
1.45 1.30
1.37 1.37
at rate of 14.6 ml./min. at rate of 7.3 ml./min. 62 1.25 90 1.81
Saline Infusion
__
26 35
31 23
27 27
20 45
30 45
30 32 30
21
27 39
‘XI Fract. Reab. ~~ ~__
-.
-13 -22
-3 -23
+29 -10
-26 +15
% Change Fract. Reab. ..~ _
7b 8b
5b 6b
3b 4b
lb 2b
7a 8a
4a 5a 6a
3a
la 2a
T.F. St3IlWlC Number ~~ -
-7% Mean ~~___ _ .._.___~ * Initial dog weight: 14.8 Kg. Day of experiment: 14.6 Kg. P,, 130 mEq./L. U 08111=urine osmolality mOsm/Gm. H,O; POR,,,=plasma osmolality mOsm/Gm. H,O; P inulin=plasma inulin concentration; C inulin=inulin clearance; TF inulin=concentration of inulin in tubule fluid: % fract. reab.= per cent of glomerular filtrate reabsorbed to point of micropuncture.
66-104 107 110 111 113 116 119 121 124 126 128 132 134 141 149
2 6 7 12 15 16 22 27 30 31 37 45 46-66
(min.)
yP$
Table
294
DAVIS
ET AL.
was done in Groups I and II. Isotonic saline was then infused at a rate of 0.5 ml./Kg. for the remainder of the experiment. Recollection of tubule fluid samples and urine for clearances were obtained as in the first two groups. In the fourth group of six additional dogs, the volume of extracellular fluid was estimated from the volume of distribution of 500 mg. of inulin given by intravenous injection before and after the induction of hyponatremia. Correction was made for loss of inulin in the urine. In all animals venous blood samples were collected at the midpoint of each urine collection period from an indwelling femoral vein catheter; urine was collected through ureteral catheters. The concentration of inulin in tubule fluid (TF In) and plasma (P In) samples was determined. The tubule fluid to plasma inulin concentration ratios (TF/P In) were calculated by interpolating the observed plasma inulin concentrations to the time of collection of the tubule fluid. Fractional reabsorption of glomerular filtrate to the point of micropuncture was calculated from the formula l-(P/TF In). For statistical evaluation, the mean change in fractional reabsorption, obtained from three to nine tubules in each dog, was considered as a single observation. Inulin was measured in tubule fluid by the microfluorometric method of Vurek and Pegram,? and in plasma and urine by the anthrone method of Fiihr et al.8 Sodium in plasma and urine was determined by flame photometry using a lithium internal standard. Plasma and urine osmolalities were measured with a Bowman osmometer. RESULTS
In the 7 normonatremic dogs, average sodium concentration 144 mEq./L., 43 samples of fluid from proximal tubules were obtained during hydropenia. After infusion of saline, fluid was collected from the same segments. The concentration ratios of inulin in tubule fluid to that in plasma (TF/P) of these 43 paired samples are plotted in Fig. 1. The mean ratio of the (TF/P) during saline loading to that in hydropenia was 0.825 + 0.065. The mean decrease in fractional sodium reabsorption calculated from these (TF/P) inulin ratios was 48.1 per cent -c 3.63 per cent. Sodium excretion rose from a mean of 7.3 f SE 1.78 pEq./min. during the control periods to a mean of 289 + SE 45.9 pEq./ 2.5 -
NORMAL f
ISOTONIC
2.0-
DOGS SALINE
HYOROPENIA
Fig. I.-Proximal tubule sodium reabsorption. Points represent paired collections of proximal tubule fluid with value of (TF/P) inulin during hydropenia plotted against value after infusion. Solid line indicates no change; broken line indicates mean Effect of infusion of isotonic saline change from control to experimental collections. to normal dogs.
EXTRACELLULAR
FLUID
VOLUME
295
EXPANSION
.0HYPONATREMIC HYPOTONIC
DOGS SALINE
A = MEAN
1.0
I. 5
2.0 1111111 Ihl
2.5
HYDROPENIA
Fig. 2.-Proximal tubule sodium reabsorption. Points represent paired collections of proximal tubule fluid with value of (TF/P) inulin during hydropenia plotted against value after infusion. Solid line indicates no change; broken line indicates mean change from control to experimental collections. Effect of infusion of hypotonic saline to hyponatremic dogs.
min. during experimental periods. The urine volume increased from 0.20 r+ SE 0.019 ml./min. to 2.6 rf: SE 0.15 ml./min. after the saline infusion. The mean inulin clearance was 58.2 & SE 5.0 ml./min. during control and 68.4 Z!XSE 5.8 ml./min. in the experimental periods. The mean weight of the dogs before induction of hyponatremia was 14.43 +SE 0.53 Kg., the mean weight on the day of the experiment was 14.40 + SE 0.52. The change in weight of -0.03 was not significant. In the seven hyponatremic dogs, average sodium concentration 126 mEq./L., 38 pairs of tubule fluid samples were obtained. These data are plotted in Fig. 2. The mean ratio of the (TF/P) inulin after the saline to that during hydropenia was 0.915 + SE 0.022. This represents a depression of fractional sodium reabsorption by the proximal tubule of 19.4 per cent rt SE 7.45 per cent, a significantly smaller decrease in fractional reabsorption than was measured in the normal dogs (p < 0.005; see Table 2). Sodium excretion increased from 3.1 k SE 0.87 pEq./min. during hydropenia to 63.2 t SE 23.0 pEq./min. after saline. This rise is significantly less than that observed in the normonatremic dogs (p < 0.0 1) . Urine flow increased from 0.30 2 SE 0.047 ml. to 3.53 -t SE 0.88 ml./min. This increase is not significantly different from the increase noted in the normal dogs. Inulin clearance increased from 66.7 * 5.77 ml./min. to 71 f 5.68 ml./ min. As shown in Table 3, there was no significant change in the plasma osmolality following the infusions; however, urine osmolality changed from hypertonic to hypotonic in each experiment. The data from 42 pairs of samples obtained from six hyponatremic dogs given hypertonic saline are plotted in Fig. 3. The mean ratio of the TF/P In after
296
DAVIS ET AL.
Table 2.-Mean
per Cent of Proximal Na Reabsorption Following Saline Infusion
Normal Dogs Isotonic Saline n tubules
(5)
-49 -59 -59 -37 -45 -52 -36
(7) (8) (6) (6) (4) (7)
Hyponatremic Dogs Hypertonic Saline n tubules
-10 -13 -31 -59 -15 -1 -7
-64 -22 -51 -55 -35 -64
(5) (5) (3) (6) (7) (4) (8)
- 19.4 k 7.47 7
-48.1 SE + 3.63 7 n dogs
I
Hyponatremic Dogs Hypotonic Saline n tubules
(7) (6) (7) (9) (7) (6)
-48.5 + 5.57 6
p < 0.005
I
I
I P < 0.02 Table 3.-Effect of Infusion of Hypotouic or Hypertonic Saiine to Hyponatremic Dogs on Plasma and Urine Osmoiaiities, and Plasma Sodium Concentration
H
Dog
P mOsm/Kg.
A. Hypotonic
Sa;Ii
S
H
zfy?Ln . .
S
H
U mOsm/Kg.
S
1 2 3 4 5 6 7
278 273 275 257 261 255 277
277 270 274 260 261 250 278
132 116 130 123 125 124 131
134 123 133 128 126 124 135
700 633 347 1000 1180 490 720
61 115 229 66 94 213 130
Mean
268
267
126
129
724
130
Doe
H
B. Hypertonic
1 2 3 4 5 6 Mean
P mOsm/Kg.
Saline Infusion PN= mEq./L.
U mOsm/Kg.
S
H
S
H
270 259 275 274 263 270
287 295 322 285 286 300
132 129 134 128 124 133
142 144 157 139 142 151
359 920 131 900 1098 294
135 96 171 750 270 66
269
296
130
146
617
248
H =Hydropenia;
S
S = Saline; P = Plasma; U = Urine.
saline infusion to that during hydropenia was 0.826 k SE 0.018. The mean decrease in fractional sodium reabsorption calculated from these figures was 48.5 I+ SE 3.6 per cent. This is not significantly different from the depression following saline infusion in normal dogs. It is, however, a significantly greater depression than that noted in hyponatremic dogs (p < 0.02; Table 2). Sodium excretion rose from 61.5 k SE 4.33 pEq./min. to 204 k 140. Urine flow rate
EXTRACELLULAR
-
FLUID VOLUME
297
EXPANSION
2.5 -
u U-J
HYPONATREMIC HYPERTONIC
DOGS SALINE
A= MEAN
,NUL,N HYDROPENIA
Fig. 3.-Proximal tubule sodium reabsorption. Points represent paired collections of proximal tubule fluid with value of (TF/P) inulin during hydropenia plotted against value after infusion. Solid line indicates no change; broken line indicates mean change from control to experimental collections. Effect of infusion of hypertonic saline to hyponatremia dogs.
from 0.66 SE + 0.038 cc./min. to 4.58 rt 1.82, neither increase being significantly different from the response to saline infusion in normal dogs. The mean inulin clearance was 50.5 cc./min. during hydropenia and this rose to 54.0 cc./min. after the hypertonic saline. The hypertonic saline caused an increase in the mean plasma osmolality from 269 to 296 mOSm/Gm. Hz0 and the mean plasma sodium from 130 to 146 mEq./L. (Table 3). The results from the six additional dogs in which plasma sodium concentration, inulin space measurements, and weights were measured before and after production of chronic hyponatremia are shown in Table 4. The change in plasma sodium concentration - 14.5 -+ SE 3.3 mEq./L. was significant (p < .Ol ) . The increased
Dog
P!yna
Table
4.-E&&
Sodiim
(mEq./k)
of Production
256
148
- 108
14.5
15.0
-3 +68 +103 -112 -32
12.0 13.0 11.5 15.0 12.0
12.0 13.3 11.7 15.0 11.9
13.0 0.6
13.1 0.6
C
1
142
135
-7
2 3 4 5 6
146 143 150 147 145
120 132 139 124 136
-26 -11 -11 -23 -9
287 134 193 218 222
284 202 296 106 190
145.5 1.1
131.0 3.0
- 14.5 3.3 < .Ol
218 22
204 30
Mean tlSE
of Chronic
Inulin Spage (ml./Kgh)
__C = Control;
H = Hyponatremia.
-14 36 N.S.
Hyponatremia C
Weigz
(Kg.)
A
+o.s 0.0 +0.3 +0.2 0.0 -0.1 +0.15 .09 N.S.
298
DAVIS ET AL.
changes in inulin space, - 14 SE _+ 36 ml/Kg. .09 Kg. were not significant.
and dog weight, f0.15
I
SE
DISCUSSION
Intravenous saline was administered to both normal and chronically hyponatremic dogs. Isotonic (.85%) saline was infused into the normals. The sodium concentration of the fluid given to the hyponatremic animals was adjusted so that the solution was isotonic to the recipient. Equivalent volumes were administered to both groups but the saline caused less depression of fractional sodium reabsorption by the proximal tubule in hyponatremic than in normal dogs. The saline also resulted in a lesser increase in sodium excretion in the hyponatremic group. These findings are in accord with those of Blythe and Welt1 and Schrier et a1.2 and localize the antinatriuretic effect of hyponatremia at least in part to the proximal tubule. The similarity in body weight before and after induction of hyponatremia indicates that total body water was unchanged by the procedure. That extracellular fluid volume remained constant is supported by the finding that there was no measurable change in the inulin space with the production of hyponatremia. The variability in these measurements is such, however, that a small change in extracellular fluid volume might have gone undetected. Fractional sodium reabsorption in the proximal tubule was similar in both normal and hyponatremic dogs during hydropenia. Therefore, starting from comparable baselines expansion of the extracellular fluid volume resulted in a significantly smaller decrease in sodium reabsorption by the proximal tubule in hyponatremic dogs than in those with a normal serum sodium. When extracellular fluid volume was expanded concomitant with restoration of the serum sodium to normal, hyponatremic dogs infused with hypertonic saline, this difference was abolished. The interpretation of these data is that hyponatremia acted to decrease the response of the proximal tubule to isotonic expansion of the extracellular fluid volume. The result of this effect of hyponatremia was that the proximal tubules of the hyponatremic dogs reabsorbed a greater fraction of the glomerular filtrate after saline infusion than did the proximal tubules of the normal animals. We have previously reported experiments in which 5 per cent albumin in saline solution was first equilibrated with the blood volume of dogs resulting in dilution of the blood without changes in vascular volume and subsequently the blood volume was expanded without changes in the composition of the blood.g It was concluded that both dilution of the blood and expansion of the blood volume are independent and important factors in the decrease in sodium reabsorption by the proximal tubule following infusion of saline solution. In the present series of experiments both isotonic solutions used would be expected to result in equivalent dilution and expansion of the blood volumes. The significantly smaller decrease in sodium reabsorption by the proximal tubules of hyponatremic dogs could be related therefore to a blunted response to either dilution or expansion. The mechanism of the decreased sodium reabsorption by the proximal tubule that is induced by intravenous saline is controversial. Two major hypothesis have been advanced. One holds that the depression in fractional sodium reabsorption
299
EXTRACELLULAR FLUID VOLUME EXPANSION
I
40 FRACTIONAL S00lUM REABSORPTION BY PROXIMAL TUBULE K
30
-
20
-
10 -
+h *
Ii
H
DE
DE
0 HYPONATREMIC
NORMONATREMIC
H
D
DE
NORMONATREMIC
DOGS
Fig. 4.-Fractional reabsorption +-1 SE by proximal tubule of normonatremic and hyponatremic dogs during continued hydropenia (H) and following dilution alone (D) or dilution and expansion (DE) of blood volume.
is mediated by an altered activity of an unknown hormone. The other suggests that the effect is mediated by a change in the physico-chemical properties of the blood perfusing the proximal tubules. The increase in sodium excretion of recipient dogs in cross circulation experiments in which the donor dog was saline loaded supports the hypothesis that infusion of saline results in a change in a humoral factor.lO-‘” Rector et al .13 have presented evidence for a humoral agent which decreases sodium reabsorption by the proximal tubule of the rat following extracellular fluid volume expansion. However, since Schrier et al.l* have been unable to confirm these results, the existence of such a hormone remains speculative. It would be expected that changes in physical factors, such as the colloid osmotic pressure in peritubular capillaries, which are thought to be responsible for the decreased proximal reabsorption during dilution would have been similar in hyponatremic and normal dogs. If that is true, the hyponatremic dogs might have responded to dilution but not expansion of the extracellular fluid volume. Support for this suggestion is found in Fig. 4 where the effects of dilution and expansion of the blood volume from the dogs previously reported9 is compared to the data obtained in these experiments. Dilution of the blood volume without expansion resulted in a depression of fractional sodium reabsorption similar to that measured in the hyponatremic dogs that received an isotonic infusion. The results of both expansion and dilution were similar to the response of the normal dogs in these experiments. ACKNOWLEDGMENT We are grateful for the invaluable technical assistance of Nordica Green and Marilyn J. Walter.
REFERENCES 1. Blythe, W. B., and Welt, L. G.: Plasma sodium concentrations and urinary sodium excretion. Trans. Ass. Amer. Physicians 78: 90-96, 1965. 2. Schrier, R. W., McDonald, K. M., Marshall, R. A., and Lauler, D. P.: Absence of natriuretic response to acute hypotonic intravascular volume expansion. Clin. Sci. 34: 57-72, 1968. 3. -, Fein, R. L., McNeil, J. S., and Cirksena, W. J.: Influence of interstitial fluid
volume expansion and plasma sodium concentration on the natriuretic response to volume expansion in dogs. Clin. Sci. 36: 371-385, 1969. 4. Dirks, J. H., Cirksena, W. J., and Berliner, R. W.: The effect of saline infusion on sodium reabsorption by the proximal tubule of the dog. J. Clin. Invest. 44: 1160-1170, 1965. 5. Howards, S. S., Davis, B. B., Knox, F. G., Wright, F. S., and Berliner, R. W.: De-
300 pression of fractional sodium reabsorption by the proximal tubule of the dog without sodium diuresis. I. Clin. Invest. 47:15161572, 1968. 6. Davis, B. B., Knox, F. G., and Berliner, R. W.: Effect of vasopressin on proximal tubule sodium reabsorption in the dog. Amer. J. Physiol. 212: 1361-1364, 1967. 7. Vurek, G., and Pegram, S.: Fluorometric method for the determination of nanogram quantities of inulin. Anal. Biochem. 16: 409-419, 1966. 8. Fiihr, J., Kaczmarcyzk, J., and Kruttgen, D. : Eine Einfache colorimetrische Methode zur Inulinbestimmung fur Nierenclearance-Untersuchungen bei Stoffwechselgesunden und Diabetickern. Clin. Wochschr. 33~729-730, 1955. 9. Knox, F. G., Howards, S. S., Wright, F. S., Davis, B. B., and Berliner, R. W.: Effect of dilution and expansion of blood volume on proximal sodium reabsorption. Amer. J. Physiol. 215:1041-1048, 1968. 10. Johnston, C. I., and Davis, J. 0.: Evidence from cross circulation studies for a humoral mechanism in the natriuresis of
DAVIS
ET AL.
saline loading. Proc. Sot. Exp. Biol. Med. 121: 1058-1063, 1966. 11. DeWardener, H. E., Mills, I. H., Clapham, W. F., and Hayter, C. J.: Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin. Sci. 21:249258, 1961. 12. Lichardus, B., and Pearce, J. W.: Evidence for a humoral natriuretic factor released by blood volume expansion. Nature 209:407-409, 1966. 13. Rector, F. C., Martinez-Maldonado, M., Kurtzman, N. A., Sellman, J. C., Oerther, F., and Seldin, D. W.: Demonstration of a hormonal inhibitor of proximal tubular reabsorption during expansion of extracellular volume with isotonic saline. J. Clin. Invest. 47:761-773, 1968. 14. Schrier, R. W., Verroust, P. J., DeWardener, H. E., Holzgreve, H., Brenner, B. M., Wright, F. S., Bennett, C. M., Keimowitz, R. I., and Berliner, R. W.: Failure to demonstrate a humoral inhibitor of proximal sodium reabsorption. J. Clin. Invest. 48: 1107, 1969.