POsrGRAD. MED. J. (I964), 40, 317

THE MECHANISMS OF URINARYCONCENTRATION AND DILUTION AND THEACTION OF THE ANTIDIURETIC HORMONE

A Review of the Literature and Modification of the CountercurrentHypothesis Based on Electron Microscopic Studies

MOHAMMAD SADEK SABOUR, D.M., M.R.C.P.E., M.R.C.P., PH.D.Department of Internal Medicine and the Renal Clinic, Ain-Shams University, Abbassia, Cairo, Egypt

MORE than a century ago, Claude Bernard firstpointed out that the true medium in which we liveis neither air nor water, but the fluid that bathesall the tissue cells. The osmotic pressure of thisextracellular fluid, and presumably of the cellsthey bathe, is carefully guarded at values of 280 to300 mOsm./kg. water. The cellular milieu of thehuman kidney, however, must somehow copewith the fluid contents within its tubules, theosmolality of which ranges from under 50 to overI,300 mOsm./kg. water, between the extremes ofmaximum diuresis and antidiuresis that is fromroughly one-sixth to over four times the osmoticpressure of the plasma.The flexibility of renal function has understand-

ably been subjected to considerable experimentalscrutiny and various conclusions have beendeduced and formulated into hypotheses toexplain the mechanisms of dilution and concentra-tion of the urine. In reviewing the historicaldevelopment of the various theories it is interestingto note how often the concentrating function ofthe kidney has been linked to the loop of Henle.Such a relationship was suggested in I909 byPeter who noted a correlation between the maximalconcentrations of the urine achieved by variousmammals and the lengths of the thin segment ofthe loop of Henle in their kidneys. In 1927 Cranepointed out that only mammals and birds can formconcentrated urine and that it is only in thesephyla that thin segments of the loops of Henleoccur. Closely related to this observation werethe experiments of Burgess, Harvey and Marshall inI933 which showed that it was only in birds andmammals that antidiuretic hormone increased thetubular reabsorption of water. In fact, on thebasis of these experiments Burgess and his co-workers were led to formulate the almost pro-phetic hypothesis that the urine was concentratedA paper given at the Ciba Scientific Bureau, Cairo,

on March 22, I963, in Seminar on 'Recent Developmentsin Renal Function and Disease'.

in the loop of Henle and that the antidiuretichormone acted upon this segment of the nephron.

This hypothesis, however, seemed to be in-validated by the classic studies of Walker, Bott,Oliver and MacDowell in I94I, which demon-strated that the tubular urine obtained by micro-puncture from the distal convolution of the nephronof the rat was at most isosmotic and was certainlynot hyperosmotic as would be expected if thefinal concentrating operation occurred in the loopof Henle. An alternative mechanism for concen-trating the urine was then suggested by HomerSmith and his co-workers Wesson and Anslow(Smith, 1951, I952; Wesson and Anslow, 1952;Wesson, Anslow and Smith, I948); until quiterecently this was the most popular and the mostplausible theory of the mechanisms of urinaryconcentration and dilution.

Utilizing micropuncture and microanalytic tech-niques, Walker and his colleagues (I94I) estab-lished that the fluid in Bowman's capsule was anultrafiltrate with respect to osmotic pressure,electrical conductivity, pH, glucose, creatinineand electrolytes. At the end of the proximaltubule:

(a) All the glucose and phosphate of theglomerular filtrate had been reabsorbed.

(b) The volume of the glomerular filtrate hadbeen reduced by 80%.

(c) The creatinine concentration had increasedalmost fivefold.

(d) The total osmotic pressure remained equalto that of the plasma.

Since creatinine is neither secreted nor re-absorbed from the tubules, intraluminal concen-tration of this substance can be achieved only byremoval of water. Net osmotic activity, however,did not change; reabsorption of water, therefore,must have been accompanied by an amount ofsolute in such proportion as to maintain isoton-icity between the reabsorbate, the tubular fluid andthe plasma. Smith and his co-workers (Wesson and

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Anslow, 1952; Wesson, Anslow and Smith, 1948;Smith, 1947; Wesson and Anslow, 1948) by aseries of ingenious experiments, proved thatsodium chloride is the main solute which is activelyreabsorbed by the proximal tubules, carrying withit an isosmotic amount of water. The net accom-plishment of operations within the proximaltubules is to return to the circulation the bulk ofthe filtered water and solutes. This process of'routine conservation' reduces the volume of theglomerular filtrate by 80 to 85%.

Smith and his co-workers discarded the viewthat the loop of Henle was the site of urinaryconcentration. The previous finding by Walkerand others (I94I) in three punctures of the distaltubule of concentrating rat kidneys that the fluidtherein may be somewhat hypotonic to plasma wasthought to indicate that the filtrate that has alreadytraversed the loop of Henle is not concentratedwithin the loop. According to Smith's theory, theactive process of primary movement of sodium inthe proximal tubule might slightly outstrip the.passive reabsorption of water, rendering the fluidhypotonic; the thin limb composed of epitheliumpermeable to water would allow diffusion of waterso that isotonicity could be re-established.The processes of glomerular filtration, proximal

reabsorption and equilibration in the loop ofHenle, resulting in an isosmotic fluid, seem to befixed and, in an osmotic sense, unselective. Tothe distal tubule and collecting duct, therefore, inSmith's hypothesis, are detailed the discriminatorymechanisms which determine the ultimate concen-tration or dilution of the urine, an operation whichSmith calls 'facultative reabsorption', mediatedlargely by the influence of antidiuretic hormone(ADH) of the neurohypophysis; and implying,unlike the 'obligatory reabsorption' in the proximalsystem, that movements of solute and water arenot always isosmotic. The isosmotic fluid in thedistal system can be rendered hypotonic by theactive removal of solute without water from thetubular fluid. The epithelium of the distal tubulein this theory, in the absence of any activity ofADH, i.e. in water diuresis or in diabetes insipidus,is virtually waterproof and so water is restrainedfrom following in the wake of the actively trans-ported sodium. For the first time, then, water isretained in the tubular lumen without beingcompletely obligated by an equivalent amount ofan osmotically-active solute.Under conditions of dehydration, or under the

influence of ADH, to elaborate a hypertonie urinefrom an isotonic filtrate there must be an abstrac-tion of water without solute. Smith preferred tolocate the concentrating site in the collecting ducts.Production of hypertonic urine, therefore, may beconceived as occurring in two phases. The first,

an 'isosmotic-making phase', is the passive diffusionof osmotically-free water liberated by the activereabsorption of sodium, under direct control ofADH, to raise its concentration to that of theplasma. The second phase, the 'hyperosmotic-making process', is the further reabsorption of anadditional amount of pure water from this residualisotonic fluid, rendering the tubular contentshypertonic to plasma. It is apparent that themovement of water during this latter operationmust take place against an osmotic gradient.Since there seemed to be no reasonable alternative,this final abstraction of water necessary to producehyperosmotic urine was thought to involve anactive transport of water.

This hypothesis of Smith and his colleagueswas widely accepted and stimulated a great deal ofclinical and physiological work. The hypothesis,however, postulated the active transport of waterand this posed considerable conceptual problems.The favourite model for active transport processesgenerally consists of a biochemical pump whichhandles molecules or ions of the transportedsubstances one by one and which is driven bymetabolic forces. With water, the energy consump-tion of the hypothetical pump would exceed byseveral orders of magnitude that predicted fromthe rate of oxygen consumption.

In 1955, Brodsky, Rehm, Dennis and Millerdemonstrated the thermodynamic impossibility ofthe theory of active water reabsorption. Thisgroup concluded that the energy required toproduce a concentrated urine by active water re-absorption would be I,OOO times the capability ofthe tubular cells. Clearly it was time to search foranother mechanism to concentrate the urine;fortunately, such a mechanism had already beensuggested.The Counter-current Hypothesis

In 1951, a bold new theory had been suggestedby Wirz, Hargitay and Kuhn, all of the Universityof Basel to explain the mechanism whereby thekidney concentrates the urine. These investigatorssaw in the structural arrangement of the loop ofHenle the possibility of the operation of a counter-current multiplier system.The basic feature of a counter-current system

is that the juxtaposition of two streams moving inopposite directions multiplies small exchanges ofenergy or material between them. The renalmedulla has a unique spatial configuration as aresult of the anatomical orientation of the structureswithin it, which provides a virtual battery ofcounter-current systems arranged in parallel(Fig. i). The descending and ascending limbs ofthe loops of Henle constitute one such system, andthe arterial and venous limbs of the vasa recta,

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June 1964 SABOUR: Mechanisms of Urinary Concentration 319

teterIofrular

Cortex

··!~,!~Outer Zoneof Medulla

I-nner. Zone.,of Med-uia·

PaVila

FIG. i.-Diagram showing a super-ficial cortical, a juxtamedullaryand an intermediate nephron.Note that the medulla consistsof a battery of counter-currentsystems derived from the loopsof Henle and the vasa recta ofthe juxtamedullary nephrons.

intermingling with and parallel to the loops ofHenle, form another. Hargitay and Kuhn (I95I)suggested that the mechanism of concentration ofthe urine is based on the presence of a long tubethat bends back on itself, so that flows in the twolimbs are opposite in direction (hairpin-counter-current), combined with some active process thatcreates a small concentration-difference betweenthe two limbs, the descending being slightlyhypertonic to the ascending.

This initial concentration-difference betweenthe two limbs could be created if sodium wereactively transported from the lumen ofthe ascendinglimb to the interstitial tissue by the tubular cells,and water were to move passively from thelumen of the descending limb as the result of theslight hypertonicity of the interstitial tissuemaintained in this way. These movements ofsodium and water will maintain the concentrationof the contents of the descending limb higher thanthose of the ascending: as the former move roundto replace the latter this effect will be continuously

multiplied until it produces high concentrations inboth limbs.

Continuous action of such a mechanism willultimately result in a steady state; the fluidwithin the system will become progressively moreconcentrated towards the hairpin-bend and re-diluted on its way back up the ascending limb,resulting in an osmotic stratification along the longaxis of Henle's loop. The result of the counter-current mechanism in the loops would be a zoneof increasing hypertonicity towards the tip of therenal papilla. Concentration of the final urine wasthen considered to occur in the collecting ductsthrough a passive movement of water from thefluid in them as they traverse the progressivelymore hypertonic medullary interstitial fluid. Thatthe final concentration of the urine occurs in thecollecting ducts is in accord with the classicaltheory of Smith: the disagreement concerns themechanism by which water is removed and the roleplayed by the loop of Henle.

Experimental evidence for this theory was

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320 POSTGRADUATE MEDICAL JOURNAL June 1964

CONCENTRATING NEPHRON ACte sodium nport DILUTING NEPHRON- Passuve water transport'*:Ureo tronsport ·.

-o> CORTEX -

0'0' -' ". "

'90.

: - '. OU ED

--- Si- - -I I;;g *s -

FIG. 2.-Diagram representing the most widely accepted version of the counter-current mechanism as it is believed tooperate in a nephron with a long loop during the processes of concentration and dilution of the urine; this is basedlargely on the views of Wirz (1957) and Gottschalk and Mylle (I959). The numbers represent hypotheticalosmolality values. No quantitative significance is to be attached to the number or thickness of the arrows and onlynet movements are indicated. The active sodium transport by the epithelium of the collecting tubule is basedon the work of Hilger, Klumper and Ullrich (1958), and the urea transport on the work of Lassiter, Gottschalk andMylle (1961). Note that in this hypothesis, the distal convoluted and collecting tubules are considered to beimpermeable to the passive movement of water in the absence of the antidiuretic hormone while the counter-current mechanism proceeds equally in both the concentrating and the diluting nephron.

presented in 1951 by Wirz, Hargitay and Kuhn.They showed by microcryoscopy on 3o0,-thicktissue slices cut perpendicularly to the axis of thepapilla, that the osmotic pressure of the fluid in thecortical tubules is the same as that of the bloodand that it increases in the medulla continuouslyto the tip of the papilla. This approach revealedthe kidney to be composed of concentric shells,each of uniform osmotic concentration.Another valuable approach has been afforded

by microanalysis of cortical and medullary slicesfor specific solutes. Ullrich and co-workers(Ullrich, Drenckhahn and Jarausch, I955; Ullrichand Jarausch, 1956) have shown in hydropenicdogs that the concentration of sodium increasesprogressively from the cortex to the tip of thepapilla.A third line of evidence consists of measurement

of the osmolality of fluid obtained by micro-puncture. It was demonstrated that fluidobtained by micropuncture from the proximalconvolution was isosmotic with peripheral plasma,that the fluid obtained from the loop bend (Gotts-chalk and Mylle, 1959) or superficial capillaries ofthe papilla (Wirz, 1953) in hydropenic animals wasmarkedly hyperosmotic, while the fluid leaving

the loop, in the early part of the distal convolution,was hyposmotic (Gottschalk and Mylle, I959;Wirz, I956).The most attractive feature of the counter-

current hypothesis is that it dispenses with theactive transport of water molecules, the entireoperation being mediated by the active transportof sodium, a process that to one degree or anotheris going on throughout the length of the nephron.Another attractive feature is that for the samemechanism (sodium reabsorption) to serve eitherto concentrate or to dilute the urine, only aredefinition of the locus of action of the antidiuretichormone may be required.

It is widely accepted that the action of ADH isto increase the permeability of the tubular epithel-ium to water. This action was attributed to theopening of hypothetical 'pores' which facilitatethe diffusion of water (Koefoed-Johnsen andUssing, I953; Sawyer, I957). The simplestinterpretation of these data is to suppose that thelocus of the 'pore' action of ADH extends fromthe early part of the distal convoluted tubule allthe way down the collecting ducts (Gottschalk andMylle, 1959). Hence, in the absence of ADH, all,or nearly all, the osmotically-free water generated

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June 1964 SABOUR: Mechanisms of Urinary Concentration 321

by the reabsorption of sodium in the loop of Henleand the distal segment remains to be delivered tothe collecting ducts; since these are relativelyimpermeable to water in the absence of ADH andtherefore isolated from the hyperosmotic medulla,this free water emerges from the kidney as acopious volume of dilute urine. No direct proofhas been presented by these workers for thisassumption about the site and mode of action ofADH (Smith, I959).

Renal physiologists seem to have largely ex-hausted their methods and special techniques ofinvestigation of this problem and they are unlikelyto get much more data. It seems probable that thepresent methods of microcryoscopy and micro-analysis of thin renal slices or micropuncture of therenal tubules and capillaries, however ingenious andaccurate, are unlikely to yield much more informa-tion. The renal tubules in the outer zone of themedulla, for instance, are deeply seated and in-accessible to micropuncture. Similarly, microcryo-scopy and microanalysis ofthin slices of renal tissue,though very valuable in giving an overall picture ofthe osmolality and chemical composition at a par-ticular level, are unable to discriminate between theminor differences in the adjacent tubules at thatparticular level.One of the oldest methods adopted by biologists

and research workers for the study of physiologyis to correlate function and morphology. However,the artifacts encountered in such forms of studywere so great that they obviated the use of thismethod of approach using light microscopic studies.However, at the molecular level attained by elec-tron microscopy, chemistry and morphologybecome one and the histologist and chemist meeton common ground.

Approaching the problem of the mechanisms ofurinary concentration and dilution and the site ofaction of the antidiuretic hormone by electronmicroscopic study, Sabour, MacDonald, Lambieand Robson (1964) examined the kidneys of thefollowing groups of rats by the electron microscopeat the height of diuresis or antidiuresis:

I. Hydrated rats, forcibly given 5 or 20 ml.water.

2. Dehydration experiment where rats weredehydrated for 24 or 48 hours.

3. Hydration followed by dehydration experi-ment.

4. Pitressin experiment where rats were given50 milliunits aqueous pitressin i.v. and were killedwhen the urine passed showed an osmolality above2,000 mOsm./kg.The most striking change found in this study

was marked thickening of the basement membraneof the descending limb of the loop of Henle inwater diuresis whether this limb is the pars recta

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FIG. 3.-The basal parts of two pars recta of proximaltubules with an intervening capillary. The basementmembranes are very markedly swollen. From aforcibly hydrated rat at the height of the waterdiuresis. (x 5,700).

:·:·:j:···:

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of the proximal tubule (Fig. 3) or the thin des-cending segment (Fig. 4). On the other hand, thisbasement membrane was found to be very thin; as

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FIG. 5.-Thin segment of a loop of Henle from a de-hydrated animal. The basement membrane is ofnormal thickness and the basal cell foldings aredeen nand cnomnlex (YTxrcnn,-

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·.:· iiill.:·:·· -:::::·':::: :-'::::-':::::::)i: :::: ii::illl:::::·:::'·:::·.::::;:::·..:· ::::: :I:.::i:::::;:. ;:;:: :::::.:·::: :ii:i::l:l: ··':':·i:i::. .:.:::.x:I· 8-ia',bj-,·!t.: ·,14 ::,:::::3iiI. i:i:i-::i:i ·:::::i:. :i..iii::.i·:··j:::.:::l.i:lii:::..il:: ::i::···:::'·:':':':':" :·:::·: liiii:i':'·:::·:::::.·:·:·..: ·.:::·:I· ,li:·:I:: ::ii:·-:i:i..·I·i'::i'··:I ·:·' :::·;::::::::_.::::::::·..:. ". ··.· i.:.::i::··.·':::::: i::· i':· ·'il· ·:II' ·::· ......::ii:·iii::::iiii:.ii:.:':'':::::;I'I:i:.:::' ;·: ::: .':: '::.· ,:. ,, ,j···i:i.··.·::. ::: :.,.::i· -:::::·:·:·:·i:: :I:: ·::B:::::·'::A··:···::;..: .... ..::. ....: .··.:..lir:.::

··::: ··:···:.:ii:iii:::::il.:::·:·:· 1:1:1-:·:·:··:: ::: .:: ..'''::' ·::· ·I·:·I:i''· ·:::: :':·: ::::::·:: .:::: :··::::::i :::::·· :::·::::, ·:::::: ji:i'' :····:· .·:·. ·:·:···:·.:.:.:. ·:··· ··· :::··:.::'ij,liii.I.'''::: ::i:. ...:.:' :':'' '''''' '''' .·:·:·.··:·· j·j::. :i:i::i::·:·: :: :i::::. i: .: ::::: .·:: ·::. ·.· ·:::::ji::.:::: :::·:::::;:::: i::iI'... -:..i:i:iii:..iiiii:i.ii·;:.

9iii:·:iiii.iiii,,:i:i .::i ;,:.. .::.;..; :·.· :·..;:.. .,...: ;..:.:.:..i;...,. :·-.···-· ·c.·j..;i:;·;:·l:·...;·...P;.:·;*;· ·.·. :.:.·:;.:-::::'.;i:::·i..·r;?j;.·;-:·;. .....;.i·i:j:I#lj. :::· .:·:::- :::::: :::::·)) !lii::·::iiliiiiii::::··iit·:::.:::.;i:8:·;..·. ·-·:··- ··· ·'·::·:::::: ··:- ·:

FIG. 6.-The basement membrane of this thin segmentfrom a pitressin experiment is of normal thickness.( x 6,oo00).

thin as the basement membrane of the rest of thenephron, in dehydrated animals and in animals towhom pitressin has been administered (Figs. 5 and6). The collecting tubules did not show openingup of 'pores' or disappearance of the intercellularcement in dehydrated animals or those givenADH as claimed by Ginetsinskii from light-microscopic studies (Ginetsinskii, 1958). On thecontrary, at the height of the water duresis lateralseparation between adjacent collecting tubule cellsin the inner zone of the medulla was quiteapparent (Fig. 7).From the data obtained by micropuncture,

!~::~.r. ....~:.::::~:i:I:t:l:.:i:::::::::I:.·::::::....::::::.:...: p..::.~ .·:i':iY:~:.:..:~

'''':"::Bi:i-::~i:j~i'.~-': .l!~i:~·::·:.·: ·i::..~: i~·J ',~'."--'-'~: :li~:~...-:r.-..--...:::~::··:::--.:..·.;:.:...-..........::~..........i~il ~:..: ~.-:~.:.~::~:.:':~.;... ~:..::~: '':..:d:::X-::!.'.::~.~t: ~~.

.:,:~.:~!..~

- '..:··:i;:·:i:.:. .,,s: i:'.-.-::: ~,:::::,:·::::: i'!.!!.,~.: .....'~ ..:~:-.::'.i'::::~-~.'::. .'.::i :.:':~'i:iai.:':'.".:r~%::~:":..::: ':'.~:.'~ . .:''::;?"''

'~::~i! !i:i:i~:~

FIG. 7.-Two adjacent cells in an inner medullarycollecting tubule showing lateral separation, butthey are still adherent at the terminal bar. At theheight of water diuresis. ( x I ,ooo).

microanalysis and microcryoscopy techniques, onecan propose numerous variations of the counter-current hypothesis that will explain the facts thatare now available. However, the electron micro-scopic studies referred to above have shown thatthe main morphological difference in the nephronbetween water diuresis and dehydration is in thebasement membrane of the descending limb ofthe loop of Henle. These results do not conflictwith the suggestion that the loop of Henle functionsas a counter-current multiplier system. Theymust, however, modify the present views in thefollowing way:-

In water diuresis, in the absence of the antidiuretichormone, the basement membrane of the descendinglimb of the loop of Henle is swollen and may becomerelatively impermeable to water. In this way itprevents the outward movement of water from thelumen into the interstitium in response to the increasedamount of sodium pumped out actively from thecontents of the ascending limb into the interstitialspace. By preventing osmotic equilibrium takingplace in this manner, the fluid in the descending limbremains isosmotic with the plasma, and the loop willno longer multiply the osmotic difference created byactive sodium transport out of the ascending limb.The interstitium at thepapilla will be only very slightlyhypertonic and very little water will diffuse backfrom the fluid flowing down the collecting ducts. Indehydration, in the presence of ADH, the basementmembrane of the descending limb of Henle's loopbecomes very thin and permeable to water and allowsthe loop to multiply the original small osmoticdifference created by the activity of the cells of theascending limb.

This modified hypothesis differs from theaccepted version of the counter-current hypo-thesis in the following important points (Fig. 8):

i. In the hypothesis suggested here, the loopof Henle acts as a counter-current multiplier in

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June I964 SABOUR: Mechanisms of Urinary Concentration 323

Active sodiumimrportCONCENTRATING NEPHRON Possive water tanspot DILUTING NEPRON

Urea transport

b .. CORTEXn ----- m----.M al og1 -

..400 400 - 400

*oo O OUTER MEDULLA 0:.4i--.. .----- ------ --I

.--

1 .' INNER MEDULLAtoo-.1 o. . i

1aN j o300 ! 01) 5.0'mi " K '·

FIG. 8.-Diagram representing the proposed hypothesis explaining the mechanisms of urinary concentration anddilution. The numbers represent hypothetical osmolality values. A semi-quantitative significance is attemptedin the number and thickness of the arrows. Note that the state of the concentrating nephron is similar to thatseen in the accepted version of the counter-current hypothesis. In this new hypothesis, the descending limb of theloop of Henle is considered to be impermeable to water (and possibly to urea) in the absence of the anti-diuretichormone and therefore the loop does not function as a counter-current multiplier in the diluting nephron.

the concentrating kidney only. In the currentlyaccepted hypothesis, the loop acts as a counter-current multiplier both in the concentrating anddiluting kidney.

2. The site of action of ADH in the currenthypothesis is on the distal and collecting tubules.In the one suggested here, ADH acts on thedescending limb of the loop of Henle.

In addition to the morphological support givenby electron microscopy to this modified hypo-thesis, the following physiological data, whichwere difficult to explain by the current hypothesis,strongly support the modification suggested:

i. Gottschalk and Mylle (1960) by puncturingthe loops of Henle and vasa recta of rats withdiabetes insipidus have found that the fluid therehas an osmolality of about 500 mOsm./kg. water.The same investigators (Gottschalk, Lassiter,Mylle, Ullrich, Schmidt-Nielson, Pehling andO'Dell, 1960) and others (Wirz, 1953) have foundthe osmolality in the loops of Henle and vasa rectablood in the concentrating kidney to be about2,200 mOsm./kg. water. If the loop of Henle wasacting as a counter-current multiplier all the timeand ADH acted on the collecting tubules, theosmolality of the fluid at the tip of the loop shouldbe about 2,200 mOsm./kg. water whether ADHwas present or absent. On the other hand, if theloop acted as a counter-current multiplier in thepresence of ADH only, the osmolality at the tip of

the papilla should be just above isotonicity inabsence of ADH, which is what has been found indiabetes-insipidus rats.

2. Ullrich and Jarausch (I956) have shownthat during water diuresis sodium and chloride areconcentrated slightly in the papilla, urea very little,and creatinine not at all. This indicates thatduring water diuresis no water diffuses out of thedescending limb of the loop of Henle into theinterstitium and so creatinine is not concentratedat all. The system does not work as a counter-current multiplier and the slight increase in theconcentration of sodium and chloride is due tothe initial effect of active sodium transport by theascending limb into the interstitium, an effectwhich is not multiplied in water diuresis. Thismay explain the results of Berliner and Davidson(i957), and del Greco and de Wardener (1956),who were able to produce urine of very slighthypertonicity (in the region of 500 mOsm./kg.) inthe absence of ADH by slowing the rate of flowthrough the system, when the urine flow in thecollecting tubules might be slow enough to allowequilibration with the medullary interstitium tooccur.The role of ADH in the current hypothesis is to

open up 'pores' in the walls of the distal and collect-ing tubules. No pores have been seen to open up inthese segments of the nephron in hydropenicanimals nor in those to which ADH has been

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24A POSTGRADUATE MEDICAL JOURNAL June I964

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:·:-:·:·..:·8·: :''.·...Li:: ir.:: ks.ila ::·.m..:::I -·:·: .:::::··:::.::. .·:·:"::'· ::16i··::: ::·::-;::1 :.::i ·:::::

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:·. ·:·;·I-:i .w

:::iana.: .·:· :ij il.li ·liisr ·:.iii ': :i::·::I:·W.b.;a38:·' '·''.:::::ii I:I;·.·:;:ie::: :.::r::

:·: .i·· .kr:4::·.1·' ::j

·.-·:

iii.li

iiii:.ii

:::n ··:-:.

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FIG. 9.-Thin segment of a loop of Henle in chronicpotassium depletion. The basement membrane isthick and fibrillar. (x 9,700).

administered, even by electron microscopy. Onthe contrary, 'pores' were seen to open up bylateral separation of adjacent cells in the papillarycollecting tubules at the height of water diuresis(Fig. 7). In the absence of ADH the basementmembrane of the descending limb of the loop ofHenle was seen to be much swollen (Figs. 3 and 4).It is suggested that in absence of ADH this base-ment membrane imbibes water, holds it intimately,swells, and thereby withstands a much higherpressure and hinders the further flow of water outof it. This suggestion as to what occurs in thebasement membrane of the descending limb ofthe loop of Henle is analogous to what has beenproved for structures of similar composition.Fessler (I960) has shown that Wharton's jelly, theRanvier bulla and a synthetic complex of waterand a high polymer of open structure and fibres,will imbibe water, swell, hold the water intimatelyin no discernible space and thereafter withstand amuch higher pressure and become resistant tofurther flow of water. The basement membraneconsists of polysaccharides, proteins, salts andwater in a gel-form in a mesh of fine fibrils (Ham,I962), and so can be expected to behave in asimilar manner to the above-mentioned structures,in water diuresis, in the absence of ADH. In thepresence of ADH, these changes in the basementmembrane do not occur or are reversed. ADHprobably affects depolymetization of the poly-saccharide macromolecules in the basementmembrane of the descending limb of the loop.

r.BlB ··I::·.: .:.:. :·:·:·'':':' :':': :::: :: ''': .:I .sS:· ::: :::::: :::::: :::: :·: ·:::: .:::'::::::I :I::··III:jiiii :::: ::::: o: ,::::::: ::: :' ::::.··: .:::...:··iii.iii0iii :::::: :::: ':':'·:'' :::'' :::::.::·.:j::· :·:::::_::;x:·:··:·:,· ·:::: :::'·:'::: :'''''::.' :. ::: ':::'·:i:' ::::·':iilii.iii·liis:::':':' :::: :·:'::·::::: #: :"':::·: :::: .:EiB:::':: .·:·: :i::::::·::::.':':' :·:· i-ni·,:I:,::::i ·:·:lil::::··· ·::·..:...:--.::;,:::: ::::: .i.:z6. :::..:i:I.··',::::.. ·:::·::::::::::_·:·:;.:.:· ::::: :·: :.·:·: i:;:·.. :·.· ·::·.r:.:. .·:: .··.·.·. ·: ::: :li:: ...:. ·.·. ...-:-::i:i::::: X.·::·.·::::-::-· ·:· -·.·. :iiii. :::::· i:l.ii:'·::i:r:··.:.:. .:···;:::.iii:.:':':.:::::'::··:I:li i·1··::':·:·:'9:':: ili.::.·u·:·*·.': '· ·.:I:::z:::ii:i··::::·:·::::::i:ii +::'l:::i: ':':::::'":jLC*r;-.·...·:·' I.,lli:iiS.''''''' :::::::'·:::::· :I:i

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·-::::::::.).ll.i;llO.TXF.:.·.:i":Ist -.E I-c.-.·-l%rsr.·?s;;·aE.c.s.p..p- Ir..;I::::W:I.R.e.Bi:'.:::BLPI:::::i:.O

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i·'·:·'.l.· .:·'.:: ·:·.·

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:::: :.: .-:: ·i:r ''':::: ...: :::::.:: ::::: :.::: :·'·':'':::::: :::·:::::5:·:;-::i:::: .::::: :·:·:··: ::.:. .:.:. ': '.':·:·: ::::: ·:·:·r::::.:.:. ·:::· .:·· ::::·.:::::-;: '" 'I"'" .' ::: :·. ·: .·:· ··:' :::.. ::·:··:::·:.. ··:I':,:::::::··.:' ;:I::::::::'·:: :· ·::'···:··I·:··-":';.iii..:iii:iil'" ·::· ..::· ··:il:,:iii'::ii: :i· ···· ···:

i:i::i::: ::·: :·:·: ii:i: :::: :ijij iiiiii''''. iiii:iI.:::::. :iii:i -::i:·:::::::i:i:::i:.:I:I ::::..: ·'::::::::1:1-1::::::::ii·I:i:,I:I..:::- ::: n i;i.··a;-8 *( :ii'rri.iii 8 i:i:i :::.,

·:· li:l::::: I:::i: ::::: ISiii

FIG. io.-Thin segment of a loop of Henle in hyper-calcaemia due to hyperparathyroidism. The base-ment membrane looks much thickened and fibrillar.(x 8,250).

The experiment of dehydration after forciblehydration has shown that the changes which occurin the nephron after a physiological dose of waterare completely reversible on subsequent dehydra-tion. On the other hand, the changes that followan unusually large water load are incompletelyreversible. This might explain the clinical observa-tion that prolonged and excessive drinking inprimary polydipsia as well as in diabetes insipiduswill gradually diminish the sensitivity of thekidney to ADH and will eventually lead to apermanent loss of the power of the kidney toconserve water (Barlow and de Wardener, I959;Kleeman, Maxwell and Witlin, I958).The study of the mechanism of urinary concen-

tration must not seem too detached from clinicalmedicine. The introduction of electron micro-scopy in the approach to this problem will add avery useful new tool that might clarify manyobscure findings. It has already contributed inthis respect. In potassium deficiency, a lesion inthe basement membrane of the descending limbof Henle's loop has been reported (MacDonald,Sabour, Lambie and Robson, 1962) (Fig. 9); andin hypercalcemia, due to hyperparathyroidism, asimilar lesion in the same area was observed(Fig. io). The investigation of the other causes ofimpairment of the power of concentration of theurine by the electron microscope can be expectedto yield rewarding results in the near future.The work presented here has been done in the

Departments of Pathology and Therapeutics, Universityof Edinburgh, Scotland, in collaboration with Drs.Mary K. MacDonald, Anne T. Lambie, and J. S.Robson, to whom I am most grateful. The views andinterpretation of the results presented in this paper,however, are entirely the responsibility of the author.

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REFERENCESBARLOW, E. D., and DE WARDENER, H. E. (1959): Compulsive Water Drinking, Quart. J. Med., 28, 235.BERLINER, R. W., and DAVIDSON, D. G. (I957): Production of Hypertonic Urine in the Absence ofPituitary Antidiuretic

Hormone, J. clin. Invest., 36, 1416.BRODSKY, W. A., REHM, W. S., DENNIS, W. H., and MILLER, D. G. (1955): Thermodynamic Analysis of the Intra-

cellular Osmotic Gradient Hypothesis of Active Water Transport, Science, 121, 302.BURGESS, W. W., HARVEY, A. M., and MARSHALL, E. K., Jr. (1933): The Site of the Antidiuretic Action of Pituitary

Extract, J. Pharmacol. exp. Ther., 49, 237.CRANE, M. M. (1927): Observations on the Function of the Frog's Kidney, Amer. J. Physiol., 81, 232.DEL GRECO, F., and DE WARDENER, H. E. (1956): The Effect on Urine Osmolality of a Transient Reduction in Glomerular

Filtration Rate and Solute Output during a 'Water' Diuresis, J. Physiol., 131, 307.FESSLER, J. H. (I960): A Structural Function of Mucopolysaccharides in Connective Tissue, Biochem. '., 76, 124.GINETSINSKII, A. G. (1958): Role of Hyaluronidase in the Reabsorption of Water in Renal Tubules. The Mechanism

of Action of the Antidiuretic Hormone, Nature, 182, I2I8.GOTTSCHALK, C. W., and MYLLE, M. (I960): Premier Congres International de Nephrologie, Evian.

, , LASSITER, W. E., ULLRICH, K. J., SCHMIDT-NIELSEN, B., PEHLING, G., and O'DELL, R. (1960): Micro-puncture Study of the Composition of Fluid from Loops of Henle and Collecting Ducts in the Rodent Papilla,Excerpta. med. (Amst.), 29, 43., (I959): Micropuncture Study of the Mammalian Urinary Concentrating Mechanism: Evidence for theCountercurrent Hypothesis, Amer. J. Physiol., 196, 927.

HAM, A. W. (1962) 'Histology', 4th Ed. London, Pitman Medical.HARGITAY, B., and KUHN, W. (I95 i): Das multiplikations-principals grundlage der harnkonzentrierung in der Niere,

Z. Electrochem., 55, 539.HILGER, 'H. H., KLUMPER, J. D., and ULLRICH, K. J. (I958): Wasserruckresorption und lonentransport durch die

Sainmelrohrzellen der Saugetierniere, Pfiiugers Arch. ges. Physiol., 267, 218.KLEEMAN, C. R., MAXWELL, M. H., and WITLIN, S. (1958): Functional Isosthenuria. An Isolated Reversible Renal

Tubular Defect, Arch. intern. Aled., o10, 1023.KOEFOED-JOHNSEN, V., and USSING, H. H. (1953): The Contributions of Diffusion and Flow to the Passage of D2O

through Living Membranes, Acta physiol. Scand., 28, 6o.LASSITER, W. E., GOTTSCHALK, C. W., and MYLLE, M. (1961): Micropuncture Study of the Net Transtubular Move-

ment of Water and Urea in Nondiuretic Mammalian Kidney, Amer. J. Physiol., 200, I 39.MACDONALD, M. K., SABOUR, M. S., LAMBIE, A. T., and ROBSON, J. S. (I962): The Nephropathy of Experimental

Potassium Depletion. An Electron Microscopic Study, Quart. J. Exp. Physiol., 47, 262.PETER, K. (I909): 'Untersuchungen uber Bau und Entwickelung der Niere'. Jena: Fischer.SABOUR, M. S., MACDONALD, M. K., LAMBIE, A. T., and ROBSON, J. S. (I964, in Press): Quart. J. exp. Physiol.SAWYER, W. H. (1957): The Antidiuretic Action of Neurohypophyseal Hormones in Amphibia. In 'The Neurohypo-

physis', Editor, Heller, H., p. 171, London: Academic Press.SMITH, H. W. (I959): The Fate of Sodium and Water in the Renal Tubules, Bull. N.Y. Acad. Med., 35, 293.

--(95I): 'The Kidney: Structure and Function in Health and Disease'. New York: Oxford University Press.(1952): Renal Excretion of Sodium and Water, Fed. Proc., II, 701.

- (I947): The Excretion of Water, Bull. N.Y. Acad. Med., 23, 177.ULLRICH, K. J., DRENCKHAHN, F. O., and JARAUSCH, K. H. (1955): Untersuchungen zum Problem der Harnkonzen-

trierung und verdunnung. Uber das osmotische Verhalten von Nierenzellen und die begleitende Elektrolytan-aufung im Nierengewebe bei verschiedenem Diuresezustanden, Pflugers Arch. ges. Physiol., 261, 62.

--, and JARAUSCH, K. H. (1956): Untersuchungen zum Problem der Harnkonzentrierung und verdunnung. Uberdie Verteilung der Elektrolyten (Na, K, Ca, Cl, anorg. phophat) Harnstoff, Aminosauren und exogenem Kreat-inin in Rinde und Mark der Hundeniere bei verschiedenen Diuresezustanden, Ibid., 262, 537.

WALKER, A. M., BOTT, P. A., OLIVER, J., and MACDOWELL, M. C. (1941): The Collection and Analysis of Fluid fromSingle Nephrons of the Mammalian Kidney, Amer. 5J. Physiol., 134, 580.

WESSON, L. G., JR., and ANSLOW, W. P., JR. (1952): Effect of Osmotic and Mercurial Diuresis on Simultaneous WaterDiuresis, Ibid, 170, 255.--,-, and SMITH, H. W. (1948): The Excretion of Strong Electrolytes, Bull. N.Y. Acad. Med., 24, 586.--,-- (1948): Excretion of Sodium and Water during Osmotic Diuresis in the Dog, Amer. J5. Physiol., 153, 465.

WIRZ, H. (1953): Der Osmotische Druck des Blutes in der Nierenpapille, Helvet. physiol. pharmacol. Acta, II, 20.(1956): Der Osmotische Druck in den corticalen Tubuli der Rattenniere, Ibid., 14, 353.

-- (I957): The Location of Antidiuretic Action in the Mammalian Kidney In 'The Neurohypophysis', Editor, Heller,H., p. 157. London: Academic Press.

- -, HARGITAY, B., and KUHN, W. (195I): Lokalization des Konzenstrierungsprozesses in der niere durch direkteKryoscopie, Helv. physiol. pharmacol. Acta, 9, 196.

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Postgrad M

ed J: first published as 10.1136/pgmj.40.464.317 on 1 June 1964. D

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