Journal of Clinical InvestigationVol. 45, No. 12, 1966

Osmolality of Distal Tubular Fluid in the Dog *JAMESR. CLAPP t ANDROSCOER. ROBINSONt

(From the Department of Medicine, Duke University Medical Center, Durham, N. C.)

Renal micropuncture observations in rodentshave demonstrated that the tubular fluid from earlyportions of the distal convoluted tubule is alwayshypotonic to plasma (1, 2). A limited number ofobservations have suggested that its hypotonicityis maintained along the entire distal tubule dur-ing water diuresis (1). In contrast, during hy-dropenia with or without a superimposed osmoticdiuresis, the distal fluid achieves osmotic equi-librium with plasma in later portions of the distaltubule (1, 2). Consequently, in the presence ofantidiuretic hormone, it has been widely acceptedthat distal tubular fluid is always isosmotic toplasma as it enters the cortical collecting ducts.

These findings in rodents have precluded a satis-factory explanation for the observation that manand dog can excrete hypotonic final urine duringcertain experimental and pathological conditionswhen antidiuretic hormone activity is presumablyadequate (3-11). These observations have beenmost apparent at high rates of urine flow and sol-ute excretion. If, as in the rat (2, 12), the tubu-lar fluid of the dog is isosmotic as it enters the col-lecting ducts under these conditions, the subse-quent excretion of hypotonic urine would requirethat the collecting ducts reabsorb solute in excessof water. Since this explanation is inconsistentwith current concepts of solute and water reab-sorption by the collecting ducts in the presence ofmaximal amounts of vasopressin (water in ex-cess of solute), some investigators (9, 13, 14) have

* Submitted for publication May 31, 1966; accepted Au-gust 19, 1966.

Supported by grants from the U. S. Public HealthService (AM 05930 and AM 07420), the AmericanHeart Association, the Life Insurance Medical ResearchFund, and the North Carolina Heart Association.

t Established Investigator of the American HeartAssociation.

Address requests for reprints to Dr. James R. Clapp,Dept. of -Medicine, Duke University Medical Center,Durham, N. C. 27706.

: Senior Investigator of the North Carolina HeartAssociation.

suggested that dog distal fluid is normally hypo-tonic rather than isotonic at the end of the distalconvolution. If so, at high rates of urine flow dur-ing osmotic diuresis, the ability of the collectingducts to abstract solute-free water might be soexceeded that hypotonic urine could be excreteddespite the presence of antidiuretic hormone.Possible relationships among rates of solute excre-

tion, vasopressin dosage, and solute-free waterreabsorption by the collecting ducts have beendiscussed fully by Orloff, Wagner, and Davidson(11).

Heretofore, direct measurements of distal fluidosmolality utilizing micropuncture techniques havenot been feasible in the dog because the distal con-

voluted tubules could not be recognized on thesurface of the kidney (15). Renal micropunctureexperiments in this species have therefore beenconfined to studies of proximal tubular function.However, in the present study, segments of thedistal convoluted tubules have been identified on

the surface of the kidney, thus permitting the ap-plication of renal micropuncture techniques to an

evaluation of the contribution of distal nephronsegments to urinary concentration and dilution innormal dogs. In contrast to earlier observations inrodents (1, 2) measurements of osmolality havedemonstrated that the tubular fluid is markedlyhypotonic along the entire length of the distal tu-bule during hydropenia as well as water diuresis.

MethodsRenal micropuncture experiments were performed on

nine healthy mongrel dogs weighing from 8.2 to 14.5 kg.Five dogs were examined during hydropenia and antidiu-resis; four animals were evaluated during water diure-sis. Anesthesia was induced by the iv administration ofPentothal sodium (25 mg per kg); thereafter small sup-plemental doses were administered as necessary. Respira-tion was maintained through a cuffed endotracheal tubevia a Harvard respirator. The ventilatory rate was ad-justed to maintain the blood Pco2 between 35 and 45 mmHg. The left kidney was exposed through a flank in-cision, and indwelling polyethylene catheters were in-serted into both foreleg veins and the ureter of the

1847

JAMES R. CLAPP AND ROSCOER. ROBINSON

experimental kidney. An additional polyethylene cathe-ter was introduced into the abdominal aorta via a fe-moral artery to the approximate level of the renalarteries.

Dogs studied during antidiuresis were deprived of wa-ter after receiving an im injection of Pitressin tannatein oil (1 U) about 16 hours before the experiment. Afterthe induction of anesthesia these animals received a singleiv injection of aqueous vasopressin (33 mUper kg). Amaintenance infusion of aqueous vasopressin in 5% dex-trose in water (50 mUper kg per hour, 0.25 ml per min-ute) was administered via a foreleg vein throughouteach experiment.

Animals examined during water diuresis were allowedfree access to water before each study. After anes-thesia, water diuresis was induced by the rapid iv ad-ministration of 2.5% dextrose in water (40 to 80 ml perkg). The initial water load was followed by a constantiv infusion of 2.5%o dextrose in water at a rate equal tothe urine flow. Water diuresis occurred within 3 to 7hours. Qualitative tests for urinary glucose were nega-tive during the entire period of water diuresis.

In each group, preparatory to micropuncture, the leftkidney was immobilized on a Lucite holder as describedpreviously (15). The administration of an initial pri-ming dose of inulin was followed by a constant infu-sion (0.3 ml per minute) of 2.5 to 5%o inulin in either5%o dextrose in water (antidiuresis experiments) or0.85%o saline (water diuresis experiments). After equili-bration of the priming dose of inulin, sequential renalclearance periods of 15 to 20 minutes' duration were ob-tained throughout each study. Heparinized samples ofarterial blood were obtained at the midpoint of eachurine collection period for the determination of the plasmainulin concentration and osmolality. Similar measure-ments were also performed on timed collections of urinefrom the experimental kidney.

A single sample of proximal fluid and two to ninesamples of distal tubular fluid were obtained from thesurface convolutions during each experiment. The tech-nique for collection of proximal fluid samples and themeans of identifying the sites of tubular puncture in thissegment of the nephron have been described previously(15). The identification of surface convolutions of thedistal tubule was facilitated by the intra-aortic injectionof 1 or 2 ml of 5% lissamine green before each distalpuncture. After its injection the dye appeared rapidlyin the peritubular circulation and proximal tubular fluid.Its passage through the lumina of the proximal convo-luted tubules was usually complete within 20 to 25 sec-onds, at which time the surface of the kidney appearedcompletely devoid of dye. Approximately 30 to 50 sec-onds later, the dye reappeared within the lumina of distalconvoluted tubules. Visible dye disappeared completelyfrom the surface of the kidney within 2 minutes afterits injection. Samples of distal fluid were collected af-ter the dye had disappeared completely from the distaltubular lumen. The total injected dose of lissaminegreen was kept as small as possible during each experi-ment, and each injection of dye was always separated by

an interval of at least 7 to 10 minutes. In general, nomore than a short segment of a single distal tubule ex-tended to the kidney surface. The number of distal tu-bules on the surface of the kidney varied greatly fromone animal to another, and none at all were observed onoccasion. The diameter of the distal tubular lumen wasusually smaller than that of the proximal tubule; oc-casionally, they were equal in size. The location of asingle distal tubule had to be noted with great care dur-ing the passage of dye because the transparency of theirepithelial lining made them difficult to see once the dyehad cleared the distal tubular lumen. However, once adistal tubule had been identified and punctured, samplescould be collected with relative ease because of a briskflow of tubular fluid even during hydropenia. Sites ofdistal tubular puncture were identified by the latex in-jection and dissection technique (16). As in the rat, thedistal convoluted tubule is defined as that segment of thedistal nephron between the macula densa and the pointat which it joins with another distal tubule to form acortical collecting duct.

Before the collection of distal fluid samples, the di-rection of flow along the tubule was first ascertained bythe injection of a droplet of colored mineral oil. Samplesof tubular fluid (approximately 0.01 Al) were then col-lected at a rate sufficient to maintain the distal positionof an injected oil droplet. Only a brief period of timewas required for the collection of these small samples.Immediately after collection, the samples were emptiedfrom the collection pipettes into a siliconized petri dishfilled with hydrated mineral oil. Duplicate aliquots(< 0.001 A0) of the sample were transferred into separatemineral oil-filled quartz capillaries (i.d. 25 AL), and theirosmolality was measured cryoscopically in an Advancednanoliter osmometer. This apparatus permitted simul-taneous measurements on the unknown sample and twostandard solutions of known osmolality (500, 100, or 50mOsmper kg of H20) . In this fashion, each of theduplicate readings was bracketed by simultaneous read-ings on two standard solutions of known osmolality.Ultramicro osmolality measurements on plasma samplesfrom each experiment averaged 98.2% of the macromeasurements on the same sample. Replicate ultramicroanalyses on different aliquots of the same sample alwaysagreed within ± 2%.

Plasma and urinary inulin concentrations were deter-mined by the anthrone method of Fuhr, Kaczmarczyk, andKruttgen. Macro measurements of the osmolality ofplasma and urine were made on an Advanced osmometer(model 64-31).

Results

Hydropenia and antidiuresis. Individual val-ues from five experiments are tabulated separatelyin Table I. Plasma and urine osmolality averaged308 + 3 and 1,627 + 553 mOsmper kg (SD),respectively. U~rine flow from the single experi-mental kidney averaged 0.10 ± 0.12 ml per min-ute (SD). The unilateral clearance of inulin

1848

OSMOLALITYOF DISTAL TUBULARFLUID

averaged 17 + 6 ml per minute (SD) for allclearance periods.

Three samples of proximal fluid and 24 samplesof distal fluid were obtained from the five dogs inthis group (Table I, Figure 1). In agreementwith previous observations in the dog during simi-lar experimental conditions (15), the ratio be-tween the osmolality of proximal fluid and that ofplasma (osmolal TF/P ratio) approximated 1.0.In contrast, the distal fluid was markedly hypos-motic to plasma along the entire length of the dis-tal convoluted tubule (Figure 1). The osmolalityof distal fluid ranged from 56 to 217 mOsmperkg, averaging 126 ± 41 mOsmper kg (SD) forall distal samples (average distal osmolal TF/Pratio, 0.41 + 0.13). Once the tubular fluid hadentered the early distal tubule, a further changein osmolality was not apparent along the length ofthe distal convoluted tubule (Figure 1). Theaverage osmolal TF/P ratio of eleven early distalsamples (0 to 50%o length) was 0.40 ± 0.18 (SD),and that of thirteen late distal samples (50 to100% length) was 0.41 + 0.11 (SD). In con-trast, the average osmolal urine to plasma ratiowas 5.3 + 2.1 (SD).

Water diuresis. The individual values fromthese four experiments are also tabulated in Ta-ble I. The plasma osmolality was lower than thatobserved during antidiuresis, averaging 279 + 3mOsmper kg (SD). Urine osmolality averaged131 + 45 mOsmper kg (SD). Urine flow fromthe experimental kidney alone averaged 1.10 ±0.36 ml per minute (SD). Inulin clearance bythe single kidney was relatively stable duringeach experiment (Table I), averaging 21 + 6 mlper minute (SD).

In agreement with previous observations (15),proximal tubular fluid was essentially isosmotic toplasma during water diuresis (average osmolalTF/P ratio of four proximal samples, 0.98 ± 0.02;Figure 1). Nineteen samples of distal fluid wereobtained (Table I, Figure 1). Again, distal fluidwas markedly dilute along the entire length of thedistal convoluted tubule. The average osmolalTF/P ratio of all distal samples was 0.24 ± 0.07(SD), a value that was significantly lower (p <0.001)than the average distal TF/P ratio duringhydropenia. The distal TF/P ratio during waterdiuresis was also lower (p < 0.001) than simul-taneous measurements of the osmolal U/P ra-

tion (average, 0.47 + 0.19). Further, tubularfluid appeared maximally dilute as it entered thedistal convolution (average osmolal TF/P ratioof eight samples of early distal fluid, 0.23 ± 0.05(SD); average osmolal TF/P ratio of elevensamples of late distal fluid, 0.24 + 0.07).

Discussion

In contrast to previous renal micropuncture ob-servations in rodents (1, 2), our data in the dogdemonstrate clearly that the tubular fluid is mark-edly hypotonic throughout the surface segmentsof the distal convoluted tubule during hydropeniaand antidiuresis. Osmotic equilibrium betweendistal tubular fluid and plasma was never achieveddespite the presence of antidiuretic hormone inadequate amounts. As a result, the tubular fluidwas always hypotonic as it entered the corticalcollecting ducts. Comparison of these findingswith previous observations in hydropenic rats (inwhich hypotonic early distal fluid becomes essen-tially isosmotic to plasma by the midportion of

I'.v

8.0

6.05.04.0

3.0

- TF/P a U/P OSMOLALITYo Hydropenia* Water Diuresis

2.0h

1.0.80

.60

.50

.40

.30

0

00

0 00

O 0 0 * O0 0

0

0 o* .0

* O00 0 00

0 0* 0

.20H

0

0-

0

8-

I

H1-

6-

p0 -

8.0

6.05.04.0

3.0

2.0

1.0.80

.60

.50

.40

.30

.20

100 25 5075 0 25 50 75 100 10%Proximal %Distal Urine

FIG. 1. OSMOLAL TF/P RATIOS FOR PROXIMAL AND

DISTAL TUBULARFLUID OF THE DOGDURING HYDROPENIAOR

WATERDIURESIS. TF/P = tubular fluid to plasma; U/P =urine to plasma.

1849

.

JAMES R. CLAPP AND ROSCOER. ROBINSON

TABLE I

Osmolality of proximal and distal tubular fluid of the dog during either antidiuresis or water diuresis*

Clear- OsmolalityDog ance Osmolal Ratiosno. period Location Cin V Plasma TF Urine TF/P U/P

%length

30 1 D35D35

3 D704 D205 D406 P40

31 1 D45

32 1 D802 D90

34 1 D75D25

2 D70D70

3 D804 D655 D50

P55

35 1 D35D90

2 D45D55

3 D30D40

4 P55D25D70D65

ml/min ml/min mOsm/kgA. Antidiuresis

17

12111411

15

109

24

21

241524

0.17

0.070.060.090.07

0.06

0.260.28

0.09

0.07

0.090.070.07

29 0.08

21 0.05

23 0.07

14 0.05

304 160101

304 195304 131304 115304 298

306 114

312 127312 190

305 89

310 101145

305 74308 56310 131

305

314 110121

314 134180

314 217111

314 29710493

162

42 1 D502 D55

D653 D60

D404 D655 D806 P50

43 1 D502 D40

D753 D305 D70

P55

46 1 D30D20

2 P45

47 1 D60D80

2 D25D80

3 D70P20

B. Water diuresis19 1.64 28413 1.53 282

17 1.30 284

18 1.00 28123 0.73 28020 0.60 280

18 1.23 27821 1.40 278

23 1.53 27917 1.27 280

19 1.07

16 0.50

28 1.10

34 0.90

33 0.63

278

278

278

277

274

106655372608072

283

53756771

120281

6135

260

5658505455

259

89 - 0.3797 0.23

0.19115 0.25

0.21136 0.28158 0.26255 1.01

114 0.19100 0.27

0.2496 0.2594 0.43

1.00

101 0.220.13

134 0.94

124 0.200.21

158 0.180.20

195 0.200.95

1,097

1,2151,4471,5011,372

1,798

609639

2,194

2,254

1,6481,3431,664

2,258

1,979

2,384

2,250

3.6

4.04.84.94.5

5.9

2.02.0

7.2

7.3

5.44.45.4

7.2

6.3

7.6

7.2

0.530.530.640.430.380.98

0.37

0.410.61

0.290.220.330.470.240.180.420.98

0.350.390.430.570.690.350.950.330.300.52

0.310.34

0.40

0.480.560.91

0.410.36

0.340.33

0.36

0.48

0.45

0.57

0.71

* Abbreviations: Cin = inulin clearance; V = urine flow; TF/P = tubular fluid to plasma; U/P = urine to plasma;D = distal; P = proximal. Values for inulin clearance, urine flow, and urine osmolality are those for the experimentalkidney alone.

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OSMOLALITYOF DISTAL TUBULARFLUID

the distal tubule) suggests strongly that the distalconvoluted tubule of the dog is much less perme-able to water than that of the rat. Thus, in thedog as opposed to the rat, that portion of the neph-ron which is relatively impermeable to water dur-ing antidiuresis must be extended to include thedistal convoluted tubule as well as the ascendinglimb of the loop of Henle. However, in addition,it must be acknowledged that more efficient soluteextraction by the distal tubular epithelium of thedog could contribute t9 the observed appearanceof hypotonic distal fluid.

If volume reduction occurs along the length ofthe distal convolution during antidiuresis, as seemslikely, the maintenance of similarly hypotonic fluidalong its entire length suggests that the materialreabsorbed which is removed by this nephron seg-ment is hyposmotic to plasma; net solute and wa-ter removal occur in proportional amounts. Theapparent absence of a progressive rise in osmo-lality demonstrates that the distal convoluted tu-bule of the dog does not contribute to the eventualosmotic concentration of the hypotonic fluid thatleaves the thick ascending limb of the loop ofHenle. The actual osmotic concentration of tu-bular fluid is an exclusive function of the collectingduct. However, it is possible that a small butphysiologically significant rise of osmolality alongthe distal convolution could have been obscuredby the scatter that individual collections from dif-ferent nephrons impart to micropuncture data.

Observations during nephron microdissection inour laboratory have shown that the collectingducts are formed by the junction of two or moresuperficial distal tubules near the surface of therenal cortex. As the collecting duct descendsthrough the outer one-half of the cortex toward therenal medulla, it is joined by an additional fouror five distal tubules from deeper nephrons. Morethan eight distal tubules rarely contribute to theformation of a single cortical collecting duct. Thenewly formed collecting duct descends straightthrough the inner one-half of the cortex where itis seldom joined by additional distal tubules. Itis important to recognize that a significant seg-ment of collecting duct passes through the renalcortex before it enters the hypertonic environ-ment of the renal medulla.

The present experiments do not identify the sitealong the collecting duct at which the tubular fluid

becomes at least transiently isosmotic to plasma asits osmotic concentration rises toward that of hy-pertonic final urine. Recognition of this site isimportant to the precise anatomic localization ofsolute-free water reabsorption (TCH2o) duringantidiuresis. If the tubular fluid becomes isos-motic to plasma within the cortical collecting duct(in which case the collecting duct fluid will pre-sumably be isosmotic to plasma as it enters the re-nal medulla), the entire length of the medullarycollecting duct would be expected to contributeto TCH20 formation in the dog .as in the rat. Inthis event, estimates of solute-free water removalshould closely parallel directional alterations ofsolute removal by the entire medullary portion ofthe ascending limb of the loop of Henle. On theother hand, if osmotic equilibrium between corti-cal collecting duct fluid and plasma is not achievedso that hypotonic fluid enters the medullary col-lecting duct, it is certain that medullary collectingduct fluid will not become isosmotic to plasma un-til it has passed an undetermined distance intothe medulla. Consequently, the entire length ofthe medullary collecting duct would not contributeto the formation of TCH2o. Nevertheless, changesin the reabsorption of solute-free water should stillprovide a qualitative index of solute removal bythe ascending limb of the loop of Henle. Presentdata in the dog do not permit a definite statementas to whether TcH2o formation is a function of theentire medullary collecting duct or only its moreterminal portions.

If osmotic equilibrium with plasma is notachieved within the cortical collecting duct, theentry of a larger volume of hypotonic fluid intothe renal medulla would also favor the subsequentreabsorption of a larger volume of dilute fluidfrom the collecting duct during the process ofurinary concentration. Such an event would notonly tend to interfere with the production of amaximally hypertonic medullary interstitium, butit might provide a partial explanation for the factthat the maximal concentrating ability of the dogis lower than that of the rat.

The finding that distal tubular fluid is even moredilute during water diuresis is of considerable in-terest. Since sodium (and its attendant anions)and urea are the major osmotically active solutes indistal tubular fluid, it is likely that the observedreduction in osmolality during water diuresis can

1851

JAMES R. CLAPP AND ROSCOER. ROBINSON

be attributed to a reduced concentration of one or

both of these solutes. If so, a reduced urea or

sodium concentration in distal fluid could occur,

either completely or in part, via at least four pos-

sible mechanisms. First, a reduced urea concen-

tration could simply reflect a diminished net ad-dition of urea to the fluid of the loop of Henlebecause of a lower urea concentration in the medul-lary interstitium. Second, a reduced sodium con-

centration in distal fluid could be the consequence

of an increased delivery of sodium to the ascendinglimb of Henle's loop if water diuresis is accom-

panied by a diminished fractional reabsorption ofsodium by the proximal tubule as suggested byprevious renal micropuncture studies (15). Alower sodium concentration could then be at-tributed to increased sodium reabsorption in excess

of water by the ascending limb of the loop of Henleand the distal tubule. Third, a reduced sodiumconcentration in the medullary interstitium sec-

ondary to an increased medullary blood flow dur-ing water diuresis might also contribute to thelower osmolality of distal fluid by reducing theconcentration gradient against which sodium re-

absorption must occur from the ascending limbof Henle's loop. Fourth, the diminished osmo-

lality of early distal fluid could reflect a diminishedpermeability to water of the tubular epithelium ofthe ascending limb of the loop of Henle and thedistal tubule. A reduction in the amount of circu-lating antidiuretic hormone would provide themost logical explanation for such a change in wa-

ter permeability. If so, the entrance of more di-lute tubular fluid into the early distal tubule dur-ing water diuresis would suggest that the site ofaction of antidiuretic hormone includes the ascend-ing limb of the loop of Henle. The present data donot establish the actual importance of any of thesefour possibilities.

The demonstration of a higher osmolality inureteral urine than that observed at the end ofthe distal tubule during water diuresis differs fromprevious observations in rodents (1). In itself,this observation suggests that water is removed inexcess of solute from the collecting duct even dur-ing water diuresis, an observation that is com-

patible with the known existence of a slightly hy-pertonic medullary interstitium during water diu-resis (17). However, since the present studieswere not carried out during maximal water diu-

resis, the higher osmolality in ureteral urine couldbe explained by an incomplete inhibition of anti-diuretic hormone secretion and its continuing ef-fect on the permeability of the collecting duct towater. Since only the surface convolutions ofthe distal tubule are accessible to micropuncture,one cannot exclude the possibility that the higherosmolality of ureteral urine may have resultedfrom the admixture of tubular fluid from deepernephrons where the osmolality may have beenhigher than that in superficial nephrons.

Regardless of the exact mechanism by whichthe dog achieves and maintains a hypotonic tu-bular fluid throughout the entire distal tubuleduring both antidiuresis and water diuresis, thedata do provide an adequate explanation for prev-ious observations on the excretion of hypotonicfinal urine at high rates of solute excretion in thepresence of adequate amounts of antidiuretic hor-mone (3-11). Under such conditions, the en-trance of a larger volume of hypotonic distal fluidinto the collecting duct could so exceed its abilityto abstract solute-free water that hypotonic finalurine would be excreted.

Summary

Surface segments of the distal convoluted tu-bule were identified in the dog, thus permittingthe first application of renal micropuncture tech-niques to an investigation of distal nephron func-tion in this species.

The osmolality of distal tubular fluid was mea-sured either during hydropenia and antidiuresisor water diuresis to evaluate the contribution ofdistal nephron segments to urinary concentrationand dilution. In contrast to previous micropunc-ture observations in rodents, the distal tubularfluid was found to be markedly dilute along theentire length of the distal tubule during both anti-diuresis (average osmolal tubular fluid to plasmaratio, 0.41 ± 0.13) and moderate water diuresis(average osmolal tubular fluid to plasma ratio,0.24 ± 0.07). Despite the presence of adequateantidiuretic hormone, osmotic equilibrium betweentubular fluid and plasma was never achieved at anysite along the distal tubule.

We conclude that the water-impermeable seg-ment of the dog nephron during antidiuresis mustinclude the ascending limb of Henle's loop and theentire distal tubule. The normal entrance of hy-

1852

OSMOLALITY OF DISTAL TUBULARFLUID

potonic tubular fluid into the cortical collectingduct provides a satisfactory explanation for previ-ous observations on the excretion of hypotonicfinal urine at high rates of solute excretion in thedog (and perhaps in man) despite the presence ofantidiuretic hormone in adequate amounts.

AcknowledgmentsThe authors are indebted to Dr. Karl Heinz Gertz for

his demonstration of the use of lissamine green and toConstance Jenkins and Glenn Sides for their technicalassistance.

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mammalian kidney in The Neurohypophysis, Pro-ceedings of the Eighth Symposium of the ColstonResearch Society, H. Heller, Ed. New York, Aca-demic Press, 1957, p. 157.

2. Gottschalk, C. W., and M. Mylle. Micropuncturestudy of the mammalian urinary concentratingmechanism: evidence for the countercurrent hy-pothesis. Amer. J. Physiol. 1959, 196, 927.

3. Levinsky, N. G., D. G. Davidson, and R. W. Berliner.Changes in urine concentration during prolongedadministration of vasopressin and water. Amer.J. Physiol. 1959, 196, 451.

4. Zak, G. A., C. Brun, and H. W. Smith. The mecha-nism of formation of osmotically concentrated urineduring the antidiuretic state. J. clin. Invest. 1954,33, 1064.

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8. Cohen, S. I., M. G. Fitzgerald, P. Fourman, W. J.Griffiths, and H. E. de Wardener. Polyuria inhyperparathyroidism. Quart. J. Med. 1957, 50, 423.

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12. Bank, N., and H. S. Aynedjian. On the mechanismof hyposthenuria in hypercalcemia. J. clin. Invest.1965, 44, 681.

13. Stein, R. M., B. H. Levitt, M. H. Goldstein, J. G.Porush, G. M. Eisner, and M. F. Levitt. The ef-fects of salt restriction on the renal concentratingoperation in normal, hydropenic man. J. clin. In-vest. 1962, 41, 2101.

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