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TRANSCRIPT
THE NATUREOF THE ACTION OF INTRAVENOUSALDOSTERONE:EVIDENCE FOR A ROLE OF THE HORMONEIN
URINARY DILUTION*
By EDMUNDH. SONNENBLICK,t PAUL J. CANNONAND JOHN H. LARAGH
(Fromi the Deparbiwitt of Medicine, College of Physicians and Surgeons, Columbia Unzivcrsitv,and the Presbyterian Hospital, New York, N. Y.)
(Submitted for publication September 15, 1960; accepted January 13, 1961)
Aldosterone increases the renal retention of so-dium chloride and promotes the excretion of po-tassium ions ( 1-11 ); but where and how the hor-mone acts in the renal tubule is poorly understood.The present study was designed to characterizefurther the mode and site of action of aldosteronein the nephron.
Information can be obtained about the site ofaction of a compound by observing its effect onfree water formation (12). According to a cur-rent concept of renal physiology (13), reabsorp-tion of solute (sodium chloride) is isosmotic inthe proximal tubule (14), whereas, in the moredistal segments, reabsorption may or may not beisosmotic (15, 16). During water diuresis (ab-sence of antidiuretic hormone) the distal reab-sorption of solute is selective, i.e., occurs withoutisosmotic amounts of water, leaving "solute-free"water behind for excretion (17). Thus, by de-termining the effect of a compound on solute ex-cretion and on free water formation during main-tained water diuresis, information may be ob-tained about its site of action (12). If aldosteronewere to act to promote isosmotic reabsorption ofsodium, the consequent reduction in solute excre-tion would be accompanied by a fall in urine flowand no change or fall in free water excretion.However, if aldosterone acts only at a more distalsite, where reabsorption is selective, the reducedsodium excretion would be accompanied by nochange in urine flow and hence, by a rise in freewater excretion.
In the present study it has been shown thataldosterone can promote abstraction of sodiumchloride exclusive of water from the tubular urine.
* This work was supported by the 'United States PublicHealth Service (Grant H-1275) and by Mrs. R. C.duPont.
t Present address: National Heart Institute, Bethesda,Md.
This action takes place in the tubule at a locusdistal to that of isosmotic reabsorption. Also, thiseffect of the hormone on sodium chloride reab-sorption appears to be dissociated from the effecton potassium excretion.
EXPERIMENTAL
Seven studies were performed on 3 subj ects withoutevidence of renal or cardiovascular disease. Four of theexperiments were done with subj ects maintained on anormal NaCl intake (4 to 6 g NaCl per day for 7days), two with the subj ects on a higher intake ofsalt for 7 days (8 to 20 g NaCl per day), and one afterdrastic salt deprivation (less than 250 mg per day for6 days). In two subjects on normal salt intake, con-trol studies of similar protocol but without administra-tion of aldosterone were performed. All experimentswere conducted in the fasting state at the same earlymorning hour with the subjects in a recumbent position.
Water diuresis was induced by having the patient in-gest about 1,500 ml of water by mouth during the hourbefore the experiment and was subsequently maintainedby intravenous infusion of 5 per cent dextrose and waterat a constant rate, at least 2 ml per minute greater thanurinary output. Adequate control periods were obtaineduntil a steady state of maximum urine flow was demon-strated, so that any subsequent change in free waterclearance could not be attributed to an increasing waterdiuresis.
When a steady state of maximum water diuresis hadbeen achieved, 1 mg of d,l-aldosterone monoacetate in10 ml of 10 per cent ethanol was administered intrave-nously.' This was followed by another 1 mg given inthe dextrose and water infusion over the next hour.After the aldosterone administration was complete, the in-fusion of dextrose and water was continued at a con-stant rate.
Blood samples were taken via an indwelling hepari-nized needle and urine collections were made with an in-dwelling catheter except in Subjects S.H. and M.U.,young males in whom adequate voluntary bladder empty-ing was possible at high rates of urine flow.
I d,l-Aldosterone monoacetate, 1,000 ltg equivalent to500 pig active d-aldosterone supplied by Ciba Pharma-ceutical Products, Summit, N. J.
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EDMUNDH. SONNENBLICK, PAUL J. CANNONANDJOHN H. LARAGH
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Plasma and urine samples were analyzed for inulinor creatinine, para-aminohippuric acid, and sodium, po-tassium and chloride, by methods previously described(12). The urinary pH was measured by a Beckman pHmeter on freshly voided samples and the urine andplasma urea concentrations were determined by a modi-fication of the urease method of Van Slyke and Cullen(18). The urine and plasma total solute concentrationwas measured with a Fiske osmometer.
Calculations. As indicated by Smith (13) and byWesson and Anslow (17), urine can be divided into twomoieties: the osmolar clearance (C0,,), the volume ofwater necessary to contain the urinary solutes in a solu-tion isosmotic with the plasma (Cosm = UVosm/Posm);and the free water clearance (CH20), the net excess or
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RESULTS
The results of these experiments are summa-
rized in Tables I, II and III and in Figures 1through 4. In Table III, a typical control proto-col is given. In Table I the complete protocol ofone experiment is given, and in Figure 1 the com-
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FIG. 1. GRAPHOF THE RESULTS OF A REPRESENTATIVE
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EDMUNDH. SONNENBLICK, PAUL J. CANNONANDJOHN H. LARAGH
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FIG. 2. EFFECT OF ALDOSTERONEON SODIUM, POTASSIUM AND CHLORIDE
EXCRETION PLOTTED AGAINST TIME IN FOUR DIFFERENT STUDIES. On theordinates the net changes from the control values are plotted for each col-lection period. The increased potassium excretion is often out of phase withthe reduction in sodium chloride excretion. Also the increase in potassiumexcretion is of considerably lesser magnitude than the reduction in sodiumchloride excretion (see text).
plete results of another experiment are depictedgraphically.
Effect on renal hemodynamics. Aldosteroneadministration produced no significant changesin either glomerular filtration rate or renal plasmaflow (Tables I and II). The subjects varied con-
siderably in their control glomerular filtration ratevalues from 102 ml per minute in M.U. to 213 mlper minute in J.O.; a similar variation was notedin the renal plasma flow. Because no consistentalteration was observed in a given patient in thesevalues after injection of the hormone, the markedchanges in solute and electrolyte excretion Ito bedescribed were not attributable to alterations in
renal hemodynamics and therefore appeared to bedue to the action of aldosterone on the renal tu-bules.
Effect on sodium and chloride excretion. Fol-lowing aldosterone administration there was a de-lay of from 20 to 60 minutes before a significantreduction in sodium chloride excretion developed.During the first 20 to 60 minutes of aldosteroneadministration variable effects were observed, andin two of the seven studies a transient increase insodium chloride excretion actually occurred in thisearly period (Figure 2, Tables I and II). Then,sodium chloride excretion was sharply reduced.The reduction reached a maximum 2 to 4 hours
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ALDOSTERONEAND URINARY DILUTION
after the aldosterone administration was begun.The depressed sodium chloride excretion did not
return toward the control values until 6 to 8 hoursafter completion of aldosterone administration.
The magnitude of the sodium retention (i.e.,/ UNaV from the control level) was the greatestin Subject J.O., who had the highest filtered loadof sodium, and was least in Subject M.U. whenhe was sodium-depleted. Presumably, in thislatter instance a maximal endogenous aldosteroneeffect was present (19, 20), and no further re-
duction in the UNaV could be produced by aldo-sterone administration. In the other six studiesthe rate of sodium excretion was reduced by from40 to 86.5 per cent of the control values, and theabsolute rate of excretion was reduced by from59 to 441 uEq per minute in these six studies.
A parallel but, in general, lesser reduction inchloride excretion was observed. In the first sixof the seven experiments (Tables I and II), chlo-ride excretion was reduced by from 21 to 66 per
cent of the control values and the decrements inrate of chloride excretion ranged from 14 to 657,uEq per minute in these six experiments. In one
experiment (J.O. on 20 g NaCl intake), the fallin chloride excretion after aldosterone actually ex-
ceeded the fall in sodium output by a considerabledegree. The reason for this single observationremains obscure.
That the changes observed in sodium and chlo-ride excretion were due to aldosterone and notto prolonged water diuresis is evident from dataobtained in two control studies. Water diuresis
was maintained for about 6 hours in two recum-
bent normal subjects. No significant changes insodium, potassium or chloride excretion were ob-served during this period. In Subject O.G.,whose protocol is presented in Table III, a stablewater diuresis was established. Osmolar clear-ance fell slightly in the first 2 hours with stabili-zation at 2.7 ml per minute while urine volumeand free water clearance (CH2O) remained rela-tively unchanged. In a second normal subject,E.S., a similar course of results prevailed with an
average urine flow of 17.6 ml per minute in thefirst 190 minutes and 16.0 ml per minute in theensuing 160 minutes. Osmolar clearance fellslightly from an average of 2.8 to 2.5 ml per min-ute during the same periods with a concomitantdecline of free water clearance from 14.8 to 13.5ml per minute. Average rates of electrolyte ex-
cretion in the first 190 minutes were: Na+, 324;KF, 106; and Cl-, 424 fuEq per minute; in thelatter 160 minutes: Na+, 411; K+, 70; and Cl-, 355,uEq per minute. These control studies are inagreement with the previous work of other in-vestigators (21-23). Altogether, these resultsappear to exclude the possibility that the observedalterations in electrolyte excretion after aldosteronewere simply a consequence of prolonged waterdiuresis.
Effect on potassium excretion and on urinarypH. In every experiment but one (M.U., salt-depleted) potassium excretion was at least transi-ently increased. The increased potassium excre-
tion often preceded the sodium chloride retention
TABLE III
Detailed protocol of a typical control experiment *
Plasma
Time V Uosn Cosm CH20 UNNV UCIC UKV Osm Na K Cl
min ml/min iAOsm/ml ml/min ml/min AEq/min IEq/min AEq/min AOsm/ml AEq/ml AEq/ml AEq/ml0 1,500 ml H20 p.o., 30 minutes before start of infusion to recumbent subject.
5% Dextrose and water infusion at 18 ml/min.+23 to 67 17.4 47 2.9 14.5 299 269 29 287 146 4.5 103+67 to 105 18.2 45 3.4 14.8 184 212 33+105 to 149 17.1 42 2.5 14.6 189 159 31+149 to 209 18.2 42 2.7 15.5 212 175 25+209 to 231 17.0 43 2.6 14.4 192 169 26+231 to 252 17.2 43 2.7 14.5 211 208 24 275 3.9 108+252 to 280 17.1 44 2.7 14.4 212 187 23+280 to 317 17.1 43 2.7 14.4 204 186 25+317 to 340 17.3 40 2.6 14.7 223 201 29+340 to +360 14.8 45 2.5 12.3 170 154 34 270 140 4.0 109
Infusion completed.
* Subject O.G., 71 kg white male on normal NaCl (approx. 6.0 g/day) intake.
907
EDMUNDH. SONNENBLICK, PAUL J. CANNONAND JOHN H. LARAGH
20O
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FIG. 3. EFFECT OF INTRAVENOUSALDOSTERONEON THE
RELATION OF OSMOLARTO FREE WATERCLEARANCEDURING
A MAINTAINED WATER DIURESIS. Plotted from controlperiods taken at the height of water diuresis and froman average of two or three experimental periods at thepeak of aldosterone effect. After aldosterone the decre-ment in osmolar clearance approaches or is equal to theincrement in free water clearance in most of the studies.The rise in free water clearance occurs in the face of a
falling osmolar clearance. The findings suggest thataldosterone abstracts sodium chloride from the urinewithout water.
and persisted during the periods of maximal so-
dium retention in only four of the seven experi-ments (Tables I and II; Figures 1 and 2).
The magnitude of the increased potassium ex-
cretion produced by aldosterone (,A UKV) was
small, ranging from + 12 to + 40 uEq per min-ute or from + 17 to + 30 per cent of the controlvalues. The increments in K+ excretion were ofsimilar degree in all studies and were of consider-ably lesser magnitude than the decrements ob-served in sodium chloride excretion.
The urinary pH was observed to fall followingaldosterone excretion (Tables I and II). Theaverage observed fall was 0.38 pH units at thepeak of the action of the hormone on total soluteexcretion.
Effect on urine flow, total solute clearance andfree water excretion. In all studies aldosteronewas administered only after the peak of water diu-resis had been achieved. The control rate of urineflow varied considerably in different subjects from10 to 45 ml per minute. These individual varia-tions in urine flow seemed to be related to thevariations in filtered load of solute (24). Afteraldosterone administration, the urine flow eitherdid not change or showed a slight decrease, such
as that which occurs in prolonged water diuresis.Therefore, the effects of aldosterone were ob-served during the descending limb of water diu-resis.
The changes in total solute excretion after aldo-sterone paralleled the changes in sodium excretionboth in magnitude and in phase. Thus, after aninitial delay of from 20 to 60 minutes a progres-sive and marked reduction in urinary osmolarityensued reaching a nadir 2 to 4 hours after thealdosterone administration. Urinary osmolarityfell to levels well below that which would be ex-pected from water diuresis alone (21-23) (20/Osm per ml). Thus, aldosterone facilitated theformation of an extremely dilute urine with an in-creased urine/plasma osmolar gradient. Theurine/plasma sodium concentration gradient in-creased markedly in like manner.
As was the case with sodium chloride excretion,the total solute excretion did not fall appreciablyin the sodium-depleted subject. However, in theother six studies total solute concentration wasreduced by from 49 to 67 per cent of controlvalues. A similar reduction in the total soluteclearance (Cos, n ) was thus observed.
Since the urine flow did not change appreciablywhile the osmolar clearance decreased markedly,an increase in free water clearance was producedby aldosterone. In the first six experiments, thefree water clearance was increased by from 1.1 to4.3 ml per minute, but in the sodium-depletedsubject, M.U., the observed changes were againslight. Since the urine flow was not rising, thecalculated increases in free water cannot be at-tributed to an increasing water diuresis, and be-cause solute excretion was falling, they cannot bethe result of an increased solute load (24). Hence,these results imply the hypertonic abstraction ofsolute from the urine, i.e., reabsorption of soluteunaccompanied by isosmotic amounts of water.
In Figure 3 the relationship between the os-molar clearance and the free water clearance be-fore and after aldosterone for all seven experi-ments is plotted. It can be seen that after aldo-sterone the fall in solute clearance (A CO--1M) isequal to, or almost equal to, the rise in free waterclearance (A C1120). In fact, in four studies ofSubjects M.U. and S.H., 1 ml of free water wasadded to the urine for each milliliter of urinecleared of solute. This implies that aldosterone
908
ALDOSTERONEAND URTINARY DILUTION
caused the virtually totally selective abstractionof solute without water from the tubular urine.
Relationship of changes in sodium and chlorideclearances to changes in osmnolar clearance. Be-cause in normal individuals sodium and chlorideare the major osmolar constituents of urine andplasma and comprise the bulk of the filtered load,these solutes are the only ones available for tu-bular reabsorption in amounts sufficient to ac-count for most of the free water generated duringwater diuresis (13). Therefore, one might as-sume that the changes in osmolar and free waterclearance produced by aldosterone were due tothe effects of the hormone on sodium (and chlo-ride) ; and, in fact, in all experiments, changes inUNaV and in Uc1V closely paralleled changes inUOSMV. Aldosterone caused alterations in sodiumand chloride excretion of such magnitude that, forthe most part, alterations in Co0M produced by thehormone can be interpreted largely in terms ofthese ions.
In an effort to relate graphically the effect ofaldosterone on sodium excretion to that on soluteexcretion (Figure 4), twice the CXa 2 caused bythe hormone has been plotted against the Cosmcaused by the aldosterone for all experiments,with a resultant linear relationship. It can beseen that the changes of Cosm and thus of freewater can be interpreted to be mainly a result ofthe effect of aldosterone on sodium (with equiva-lent anions). Hence, it can be concluded thataldosterone produced a fall in C0511, and a rise inCH2O by the abstraction of salt from a distal por-tion of the nephron.
Effect on urea excretion. In all experiments,the urea clearance declined gradually with noacute change observed following aldosterone ad-ministration. These data are not given in thetables.
DISCUSSION
A number of the observations made in thepresent study appear to confirm and extend the
2 For a A C..,. of 1 ml, 1 X Po..1 (approximately 280gOsm) was abstracted from tubular lumen. For a A CNaof 1 ml then, 1 X PNa (approximately 140 /LEq) of Na'was abstracted. Each microequivalent is an osmoticallyactive particle. In abstraction of Na+ an equivalent num-ber of anions, also osmotically active particles, was re-moved. Hence, to relate A CNa to A COSm, one must mul-tiply A CNa X 2.
findings of other investigators. Thus, the delayedonset of action (3. 7, 25-29), the lack of effect onrenal hemodynamics (2, 28, 30), the sodium andchloride retention, and the kaliuresis of aldosterone(1-13) have all been described under a varietyof experimental and clinical conditions.
In the present study a latent period of about20 to 60 minutes was observed before there wasany demonstrable effect of the hormone on so-dium excretion. The peak effect occurred in 2to 4 hours, and the action lasted for as long as 6to 8 hours after administration. In previous stud-ies of man, other investigators have also noteda similar latent period and a similar duration ofactivity (3, 7, 25). Moreover, Barger, Berlinand Tulenko (26) and Ganong, Mulrow and Hol-linger (31, 32), utilizing renal artery infusions,and Crabbe (27), utilizing an isolated toad blad-der, have also noted the delayed onset of actionof the hormone.
Mineralocorticoids such as desoxycorticosteroneand aldosterone, in short-term studies, do not ap-pear to alter renal hemodynamic function eitherin experimental animals (28-33) or in man (34-36). In the present study aldosterone did notproduce significant changes in the renal plasmaflow or in the glomerular filtration rate. There-
12
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FIG. 4. RELATIONSHIP BETWEENTHE CHANGEIN OS-MOLARCLEARANCEANDTWOTIMES THE CHANGEIN SODIUMCLEARANCEPRODUCEDBY ALDOSTERONE. Two times thedecrement in sodium clearance after aldosterone ad-ministration is plotted against the corresponding decre-ment in osmolar clearance produced by the hormone.The plot illustrates that the observed changes in osmolarclearance produced by the hormone are largely accountedfor by a similar change in sodium reabsorption withattendant anion.
909
EDMUNDH. SONNENBLICK, PAUL J. CANNONANDJOHN H. LARAGH
fore, it would appear that the observed effects onelectrolyte and water excretion are the result ofinduced alterations in the activity of tubular trans-port systems.
The intravenous administration of aldosteronecaused increased tubular reabsorption of sodiumchloride and a kaliuresis. The increased potas-sium excretion often began prior to salt retention,was relatively small in degree, and at times hadceased before the sodium retention had reachedits peak. Similar effects of aldosterone have beendescribed both during acute administration (2, 7,25, 26, 31-33) and in more prolonged balancestudies (1, 3-6, 37).
In the present study aldosterone usually causedsodium retention in excess of chloride, confirmingthe results of August and Nelson (7), who ad-ministered aldosterone intravenously with iso-tonic saline infusions. It thus appears that aldo-sterone acted to augment the active renal tubularreabsorption of sodium ion with chloride. Prece-dent for the stimulation of active sodium trans-port by aldosterone has been found in vitro byCrabbe (27), using an isolated toad bladder.
According to present concepts (38), filteredpotassium is completely reabsorbed before the po-tassium destined for excretion is secreted into thetubular urine by a distal Ki for Na+ ion exchangeprocess. However, while the kaliuresis observedin these experiments may have resulted from anacceleration of this ion exchange mechanism, con-siderable evidence indicates that the predominanteffect of aldosterone-i.e., to cause sodium andchloride reabsorption-occurred independently of,and at a different locus in the distal nephron fromthe K+ for Na+ ion exchange process. Thus, thekaliuresis in general was not temporally related tothe sodium retention, the magnitude of the kaliure-sis was far less than the sodium retention, andthe degree of kaliuresis in each subject was notrelated to the corresponding degree of sodium re-tention. Ion exchange, Na+ for K+, does not ex-plain the significant chloride retention which oc-curred concomitant with sodium reabsorption.Furthermore, ion exchange does not explain thereduced osmolarity of the urine after administra-tion of the hormone, since the observed decrementsin NaCl excretion were, in fact, accompanied bycomparable reductions in total solute excretion.
The present study demonstrates that aldosterone
can cause the abstraction of sodium chloride with-out water from the tubular urine, a finding whichhas not been reported heretofore.3 During main-tained water diuresis aldosterone caused the re-moval of sodium chloride from the urine, produc-ing a fall in osmolar clearance while urine flowwas maintained; this resulted in a concomitantrise in free water clearance. For Subjects S.H.and M.U., the relationships between solute ab-straction and free water formation were such(Figure 3) as to suggest that salt was abstracted
virtually without water. However, in Subject J.O.the increment in free water clearance was lessthan the decrement in osmolar clearance indicatingthat, in this subject, in addition to selective so-dium chloride reabsorption, some additional isos-motic reabsorption may also have been induced.
Previous studies have indicated (24, 39, 40)that, during water diuresis, there is a direct rela-tionship between solute excretion and free waterclearance, a rise in Cosm resulting in a rise in CHO-Therefore, the rise in free water clearance notedafter aldosterone may have more significance be-cause it occurred in the face of a falling solute ex-cretion. Further, because the hormone was ad-ministered only after a peak of water diuresis wasachieved, when urine flow was either stable or de-clining, the increment in free water excretion afteraldosterone administration cannot be simply theresult of an increasing water diuresis.
Because aldosterone facilitates net abstraction ofsodium chloride without water from the urine, itthus appears to act in the nephron at a locus dis-tal to the site of isosmotic reabsorption. The con-cept that free water is formed in the more distaltubular segments by selective reabsorption ofsolute without water is well supported by a numberof studies (14-17). Micropuncture studies haveshown that in the proximal tubule, sodium reab-sorption is isosmotic (14-16), while fluid fromthe early distal tubule has been found to be hypo-tonic regardless of whether the final urine ishypo-or hyperosmotic (16). Vander and co-workers, using a stop-flow technique (41), havereported that in adrenalectomized dogs, aldo-sterone increased the maximal concentration gra-
3 Dingman and associates measured the osmolar clear-ance during infusion of aldosterone. However, water di-uresis was not maintained in these studies (2).
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dient for sodium which could be developed be-tween the plasma and distal tubular urine.
Because the hormone caused a distal abstrac-tion of salt without water from the urine, an in-creased plasma/urine sodium concentration gradi-ent was formed and the urine became more dilute.The results thus suggest that aldosterone is con-cerned with the production of a hypotonic urine.Inferential support for this role of aldosterone inurinary dilution may be derived from previousstudies of electrolyte and water metabolism inadrenalectomized dogs (29, 30, 42, 43) and inpatients with adrenal or pituitary insufficiency(35, 44, 45). In this group a characteristic pic-ture is seen with low glomerular filtration, in-creased loss of sodium chloride (43), and a defi-cient ability to excrete a water load (47). Min-eralocorticoid (46) alone, while promoting saltretention, does not repair the defect in water ex-cretion. Cortisol (20, 30, 44, 46) increases the fil-tration rate, increases filtered sodium load, andallows more salt to be reabsorbed from the dis-tal tubule with the production of free water andthe excretion of a more dilute urine. However,this process is not very efficient, and although theability to excrete water is improved, this occursat the expense of substantial urinary wastage ofsodium chloride. With cortisol plus a mineralo-corticoid (35, 45), solute (i.e., NaCl) can bemore efficiently removed from the distal tubulewith both the conservation of sodium and the si-multaneous production of a potentially more hypo-tonic urine for excretion. A similar function forthe endogenous adrenal mineralocorticoid hormone,aldosterone, is implied by these data.
SUMMARY
Aldosterone (2 mg d,l-monoacetate) was givenintravenously during a maintained water diuresisto normal subjects. No significant changes inglomerular filtration rate or in renal plasma flowwere observed.
After a latent period of from 20 to 60 minutes,during which natriuresis actually occurred in twoof the seven studies, the hormone produced amarked reduction in sodium with a lesser reduc-tion in chloride excretion. The peak of this ef-fect occurred in 2 to 4 hours.
Aldosterone also increased potassium excretion,
but this effect was often temporally dissociatedfrom the major action of the hormone, which wasto cause sodium and chloride retention.
After aldosterone administration urine floweither did not change or declined slightly; theosmolar clearance was reduced, and hence the freewater clearance was increased. In some of thestudies, the decrement in osmolar clearance ap-proached or was equal to the increment in freewater clearance produced by the hormone.
The results suggest that, under circumstancesof water diuresis, aldosterone can promote netabstraction of sodium chloride from the urine,virtually without water, in a distal portion of therenal tubule.
The action of aldosterone results in the crea-tion of an increased plasma/urine sodium con-centration gradient and the formation of a morehypotonic urine. It is therefore suggested thatthe hormone plays a role in urinary dilution.
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