nh3* - dm5migu4zj3pb.cloudfront.net · journal of clinical investigation vol. 42, no. 11, 1963...

Journal of Clinical InvestigationVol. 42, No. 11, 1963

DIFFUSION OF GASESOUTOF THE DISTAL NEPHRON-SEGMENTIN MAN. I. NH3*

By KENNETHA. WOEBER,t EDWARDL. REID,4 IRIS KIEM, ANDA. GORMANHILLS

(From the Section of Metabolism, Department of Medicine, University of Miami School ofMedicine, Miami, Fla.)

(Submitted for publication March 4, 1963; accepted July 18, 1963)

Physiologic variation in rate of urine flow inmammals is ascribed principally to reabsorptionfrom distal tubules and collecting ducts of a vary-ing quantity of nearly solute-free water (1-4).Water reabsorption is brought about by bulk flowor diffusion (5-8) incited by the difference inosmotic pressure between the luminal contents andthe hypertonic fluid of the adjacent renal medul-lary interstitium, a process promoted by the ac-tion of endogenous vasopressin upon the perme-ability of the intervening membranes (7, 9).Elaboration of urine much less than maximally di-lute increases the osmolality of luminal fluid as itprogresses down the most distal portion of thenephron; the result must be to raise the diffusionpressure of all substances dissolved in luminalfluid.

Two highly diffusible substances, the gases NH3and CO2, are present in solution in luminal fluidtogether with their hydrated forms NH4OHandH2CO3, in turn in equilibrium, so as to consti-tute two buffer systems, with the ionized speciesNH4+ and HCO3-, respectively. If, as a resultof the reabsorption of solute-free water, thesegases are reabsorbed by diffusion, the existenceand magnitude of the process should be reflectedin alterations in the acid-base composition ofvoided urine no less than by changes in the rateof excretion of total ammonia and total carbon

*Work supported by U. S. Public Health Servicegraduate training grant 2A-5061 of the National In-stitute of Arthritis and Metabolic Diseases, Bethesda,Md., and by a grant during part of 1962 from theGeneral Research Funds of the University of MiamiSchool of Medicine, Miami, Fla. For reprints, addressDr. A. G. Hills, Jackson Memorial Hospital, Miami 36,Fla.

t Trainee, National Institute of Arthritis and MetabolicDiseases, 1961-62.

t Trainee, National Institute of Arthritis and MetabolicDiseases, 1962-63.

dioxide. Examination of the effects of water diu-resis in normal man upon these various param-eters has provided indirect evidence that the gasesNH3 and CO2 do in fact diffuse out of the neph-ron's lumen during antidiuresis (10-12).1 Thiscommunication presents evidence that the rate ofurinary ammonia excretion is largely determinedby factors affecting the net diffusion of the freebase NH3 into the distal portion of the nephron;the evidence further indicates that some NH3,having diffused into the lumen, subsequently dif-fuses back into the renal interstitium as a resultof reabsorption of water from the lumen.

METHODS

Experimental design. Subjects were 12 healthy adultmale volunteers whose rate of urinary flow was causedto vary between 1 ml per minute and the physiologicceiling by controlled water-drinking. Serial, timed urinecollections were made at intervals of about 12 minutesby voluntary emptying of the bladder. The individualexperiments, generally comprising 20 or more collec-tions, were begun about 8 a.m. in subjects who fastedovernight and remained upright during and for at leastan hour before the experiment. When alkaline or mark-

1 "Ammonia" means total ammonia. NH, includesNH4OHwhen ratios of the buffer species are in ques-tion, and HCO3 includes anhydrous CO, in the anala-gous context. NH4, is not considered a form of bufferedH' except that "total H`" excretion includes NH,+.

It has been inferred from experiments on toad blad-der and other membranes (5) that vasopressin pro-motes osmotic movement of water out of the nephron byhydraulic flow through pores rather than by diffusion.The facts about the toad bladder are disputed (8), andthere is no direct information about the nephron. It isunderstood that postulated movement of NH, out ofthe nephron by "diffusion" might actually be caused bysolvent drag. This is immaterial to the argument as longas NH+ is restrained.

Although the term is anatomically objectionable (13),"nephron" will refer to the structure consisting of onecollecting duct and its tributary tubules.

1689

K. A. WOEBER,E. L. REID, I. KIEM, AND A. G. HILLS

Subject H. (010)pH range 5.60-6.23

No preparation

41*

** 4 *

* *** 9

9*

20 -

2 4 6 8 l0Urine Flow mi./min.

Slope + 1.0000

12 14

FIG. 1. DATA FROM A REPRESENTATIVE EXPERIMENT AT INTERMEDIATERANGEOF URINARY PH. Treatment and range of pH, which varies withflow, are indicated at upper left. Points indicate ammonia excretion dur-ing a single collection period at the corresponding urinary flow rate; slope(lower right) represents the regression, in microequivalents per milliliter,calculated by the method of least squares as the straight line best fittingthe data, of urinary ammonia excretion on urinary flow rate.

edly acid urines were desired, they were secured bypremedication for not over 18 hours with sodium bi-carbonate solution, or with ammonium chloride in gelatincapsules. Alternating 5- and 30-minute collections havealso been made at flow rates of 2 to 5 ml per minute;diminished ammonia excretion was not seen in the longercollections, indicating that no appreciable ammonia isreabsorbed from the bladder under these conditions.

70

60Total

Ammonia1sEq. 50min.

To assure that observed variation in the excretoryrate of ammonia and other urinary constituents was relatedto flow rate and not to diurnal variation or other un-suspected factors connected with elapsing time, eachexperiment consisted of two diuretic peaks preceded andseparated, and sometimes also followed, by low-flowcollections. We took care to moderate proportionalchange in rate of flow in successive samples.

Subject H. (024)pH range 4.91-5.64

Prior oral NH4CI loading

* ** .9 } M

40+

Slopea + 0.6978

2 4 6 8 10Urine Flow ml./min.

12 14

FROM A REPRESENTATIVE EXPERIMENT AT LOWESTRANGE OFURINARY PH, See details for Figure 1.

1690

60±

Total 50-Ammonia

IEq.min. 40-

30 +

FIG. 2. DATA

DIFFUSION OF GASESOUT OF THE DISTAL NEPHRON-SEGMENTIN MAN. I. NI1f 69

Subject H. (*29)Prior oral NaHC03loading

pH range 6.95-748

* *

Slope =+ 0.1938

2 4 6 8 10 12 14

Urine Flow mi./min.

FIG. 3. DATA FROM A REPRESENTATIVEEXPERIMENT AT HIGHEST RANGEOF

URINARY PH. See details for Figure 1.

The rate of total acid excretion in the more acidurines, estimated as the sum of the excretory rates of am-

monia and titratable acid, varied little and inconsistentlywith flow (Table II). There was usually also no con-

sistent variation of total acid excretion with time, al-though a few experiments showed a definite trend, alsodiscernible in comparison of urines voided early andlate at comparable flow rates. A trend downward oftotal acid excretion with time was considered to reflecta decreasing rate of H1-secretion, related either to with-drawal of prior acid loading, or to some undetectedcause, perhaps gastric secretion of HCI. To counteractthis trend in experiments in which subjects had beenpremedicated with NH4Cl, we gave a small amount ofacid (40 /AEq per minute) as NH4CI throughout the ex-

periment in divided oral doses per period. Results were

satisfactory as judged by near-constancy of total acidexcretion throughout the experiment. Slightly greater

amounts of alkali (100 AEq per minute) were usuallygiven as NaHCO3 throughout experiments conductedupon withdrawal of alkali-loading, with satisfactoryresults.

Analytical methods. Urinary pH and Pco2 were de-termined under anaerobic conditions at 370 C immediatelyupon collection, with, respectively, a Metrohm capil-lary glass electrode 2 and the electrode of Severinghausand Bradley (14). All other analyses were made induplicate and were discarded and repeated if agreementwas unsatisfactory. Ammonia was determined promptlyby microdistillation (15) from samples acidified andchilled immediately upon collection. Creatinine content(16) of all urine samples was determined, and regressionof creatinine excretion on flow was always examined;

2 Blood parameter analyzer, Epsco, Inc., Cambridge,Mass.

,BLE I

Effect of urinary pH on slope of linear regression of urinary ammonia excretion on urinary flow rate*

Urine pH at No. of Mean slope of regression of ammonia Mean increment of ammoniamax. diuresis experiments excretion on urine flow rate excretion at maximal diuresist

iLEqlml SEM jAEq/min %<5.9 5 +1.0437 4i.1164 13.4 28.4

[8] [+1.0090] [E.1673] [12.4] [26.2]5.9 to 6.5 7 +0.9952 4.0794 12.7 45.8

[17] [+1.0286] [E.0774] [13.5] [51.2]>6.5 8 +0.2890 ±.0364 3.3 78.8

[20] [+0.3285] [±.1774] [3.9] [71-9]

* The 20 experiments represented by unbracketed figures are considered technically above reproach. Estimatesbased on these differ little from estimates in brackets, based on all 45 experiments; recognized, minor deficiencies in anumber of experiments (see footnote to Table II) were too trivial to affect the results materially.

t Derived from slope of regression and from range of flow rate in each experiment.

._ci

0 -

cs

0EE

6-

3

0

* Ah, +*** t *

*4 *

1691


Subject H. No prior pro

pH ronge 560-6 23

50r

401

30k xix

x

x

20L

E 30c,w

* 20

IV

.0; 0

o C

000

00

0&5

HI II1 II1 2 3 4 5

Urin,

there was no significant or consistent variation of cre-eporotion or pH monipulotion atinine excretion as a function of flow. Creatinine con-

(#10) tent of urine samples was used as an index of subopti-mal bladder emptying, and correction to constant meancreatinine excretion was applied to the data relating ex-cretion of ammonia and titratable acid to urinary flow

xx Xx x rate where there was appreciable random variation ofx X X x x xx apparent creatinine excretion rate, and where compari-

son of corrected and uncorrected data showed that cor-rection to constant creatinine appreciably reduced thevariance of regression on urinary flow rate of urinarytitratable acid (3 experiments) or ammonia excretion(12 experiments). Slopes of these regressions wereneither markedly nor consistently affected by such cor-rection. In the remaining experiments, no correctionwas applied to the regressions, except that any indi-vidual point was discarded if creatinine excretion devi-

° 00 °0 ated from the mean for the experiment by more than0 0000 8 20%-.

0 8 Urinary "titratable acidity" refers here to residualtitratable acid excretion (or titratable acidity minus CO2),

6 78 9 10 11 12 13 14 and was determined by titration to pH 7.4 after acidi-e Flow ml / m11 fication and aeration on urine samples diluted in vitro to

compensate for variable in vivo dilution; the end pointkTER DIURESIS ON RATE OF URI- was estimated by visual colorimetry, or by the SargentL AMMONIAAND OF TITRATABLE model D automatic recording titrator, which also gave

titration curves of urinary nonvolatile buffer.

SubjectE. Prior oral NH4CI loading

x x x x

xx

(09)

x

x x

x

x x x x xx

1 2 3 4 5 6 7 8 9 O I1 12 13 14 15 16 17Serial Sample Number

18

FIG. 5. EXPERIMENT IN A SUBJECT WITH MODERATELYACID URINE BE-FORE WATER-DRINKINGAND WITH URINARY PH DEPRESSEDBY PRIOR INGESTIONOF AMMONIUMCHLORIDE. The parallelism between urinary flow rate andpH is representative for all experiments with similar initial pH. Thegraph also illustrates general design of all experiments reported here; serialsamples along abscissa represent consecutive, quantitative urine collectionsat intervals of about 12 minutes.

1692

C

E

Co

wAI.

0EE

0c

FIG. 4. EFFECT OF WANARY EXCRETION OF TOTA:ACID. See also Table IIL

x6 .0

a 5.51-.'; x

._ I

L L

I 5

>_

DIFFUSION OF GASESOUTOF THE DISTAL NEPHRON-SEGMENTIN MAN. I. NH3

TABLE II

Effect of urinary flow rate on urinary excretion of titratable acid*

A BSlope of regression Slope of regression of

Experiment of ammonia excretion titratable acid excre- Algebraic sum ofno. pH range on urine flow rate* tion on urine flow rate* slopes A + B

10 H 5.5 to 6.1 +1.0000 -1.3025(+0.716 to 1.284) (-1.071 to - 1.534) -.3025

12 H 5.6 to 6.2 +1.0245 -0.9505(+0.190 to 1.860) (-0.017 to -1.735) +.0740

15 S 5.3 to 5.9 +0.7053 -0.9113(+0.122 to 1.288) (-0.688 to - 1.135) -.2060

22 M 5.4 to 6.3 +1.7232 -0.8784(+1.229 to 2.217) (-0.081 to -1.749) +.8448

24 H 4.8 to 5.6 +0.6978 -0.8874(+0.613 to 0.782) (-0.530 to - 1.244) -.1896

26 H 4.7 to 5.6 +1.6925 -1.6061(+0.488 to 2.897) (-1.084 to -2.128) +.0864

27 E 5.6 to 6.3 +1.4131 -0.8485(+1.096 to 1.729) (-0.591 to -1.105) +.5646

38 R 5.1 to 5.8 +0.9965 -1.1771(+0.361 to 1.632) (-0.619 to -1.735) -.1806

40 R 4.7 to 5.3 +0.7113 -0.4125(+0.394 to 1.029) (-0.094 to -0.731) +.2988

43 R 5.1 to 5.9 +1.1545 -0.8360(+0.877 to 1.432) (-0.515 to -1.157) +.3185

44 R 5.9 to 6.3 +0.9797 -1.2807(+0.273 to 1.686) (-0.709 to -1.898) -.3010

45 Z 4.9 to 5.7 +1.6583 -1.9040(+0.918 to 2.398) (-1.191 to -2.616) -.2453

Mean +.0635(-.181 to +.308)

* 97% confidence limits are given in parentheses.Data are summarized from all experiments with urinary pH not over 6.4 during maximal diuresis and with data on

the excretion of titratable acid as well as ammonia. Experiments 22, 26, and 27 were technically substandard, eitherbecause there was a small downward trend with time of the sum of ammonia and titratable acid excretion, or because ofnonuniform distribution of urine flows. If these experiments are excluded from the statistical analysis, the mean of thealgebraic sums is -0.0815, with 95% confidence limits of -.212 to +.109.

RESULTS

Effect of urinary flow rate on urinary ammonia

excretion

In 45 successive experiments, without excep-

tion, the rate of ammonia excretion has varied di-rectly with urinary flow rate. Figures 1, 2, and 3depict this relation as it was observed in threerepresentative experiments in different pH ranges.

The slope of the best-fitting straight line was

invariably positive and almost always significantlyso. A tendency was sometimes noted for the rateof increase of ammionia excretion with increasingflow to fall off as flow approached its ceiling; pro-

ducing a small upward convexity of the curves.

Transformations of the data did not approximatelinearity, so comparison was made with the

straight lines best fitting the data. Some non-uniformity may arise from this procedure and mayaffect comparison if points are not evenly dis-tributed along the abscissa; nonuniformity of thisdistribution was therefore one criterion of ex-clusion from the 20 most satisfactory experimentsanalyzed (see footnote to Table II).

Table I shows the influence of urinary pHrange on the slope of the linear regression of uri-nary ammonia excretion on urinary flow rate.Also shown is the mean increment of ammonia ex-cretion as urine flow rate rises to its physiologicalceiling (mean 13.0 ml per minute from about 1ml per minute). This increment is relativelyvery small (3.3 4Eq per minute) in the high-est range of urinary pH, and much greater inboth the intermediate and the lowest pH range

1693

K. A. WOEBER,E. L. REID, I. KIEM, AND A. G. HILL.S

Subject H (# 28)pH range 5.15-5.99

*

* * *4* 4 *

2 4 6Urine Flow

Subject R (#43)pH range 5.11-5.90

000

0

8mL /min.

10 12 l4

0

0

00

0 0

0 000 00

0

I I I I I I I I

2 4 6 8 10Urine Flow ml/min.

FIG. 6. EFFECT OF WATERDIURESIS ON URINARY FREE-BASE [NH,]. Dataare from representative experiments at lowest range of urinary pH.

( 12.7 and 13.4 FAEq per minute, respectively).The percentile increase of ammonia as flow rises,however, is greatest in alkaline and neutral urine(78.8%), where the absolute rate of ammoniaexcretion is very low, falling progressively to45.8 and 28.4Ao in moderately and very acidurine, respectively, as increasing amounts ofammonia are excreted per minute.

Effect of urinary flow rate on urinary excretionof titratable acid and on urinary pH

Titratable acidity. Figure 4 shows data froman experiment representative of those in whichmaximal urinary pH is less than 6.4. Titrat-

able acidity rises, as urine flow falls, by an

amount approximately equivalent to the decreaseof total ammonia excretion over the same range

of urine flow. Table II presents values for theslopes of the regressions of ammonia and of ti-tratable acid on flow in twelve successive experi-ments of this kind. The paired values for theslopes of the two regressions in each experimentare of similar magnitude if sign is ignored (seealgebraic sums of slopes, Table II), and no pairedvalue for any experiment differs significantlyfrom its fellow, whereas all slopes differ signifi-cantly from zero. Three experiments are re-

garded as quantitatively less reliable than the othernine (see Table II).

. .

8

Urinary

CNHo 6ju Eq.

ml. 4

x

10-3 2

lH 80x(iIu

6-

z 4'

0Iam2

W

0

12 14 16

1694

+

DIFFUSION OF GASESOUT OF THE DISTAL NEPHRON-SEGMENTIN MAN. I. NH3

Subject H (*29)pH range 7.0-7.5

0 0

00

00

00000000 000 0 0

I I I I I I II

0 2 4 6 8

Urine Flow ml./min.

10 12 14

Subject R (* 36)pH range 5.69- 6.47

0

0

0 0

0 0

0

2 4 6 8 10Urine Flow ml./min.

FIG. 7. EFFECT OF WATERDIURESIS ON URINARY FREE-BASE [NH3]. Dataare f rom experiments representative of those at intermediate and highranges of urinary pH.

Urinary pH. In all of 26 experiments whereurinary pH at high flow was below 6.6 at 370 C,[H+] of urine, like the rate of excretion of titrata-ble acid, varied as an inverse function of urinaryflow rate. Change of urinary pH, with variationof urinary flow rate between 1 to 2 ml per minuteand the ceiling, was of considerable magnitude,generally 0.5 to 1 pH unit; a typical experimentis represented in Figure 5. Observations re-

corded from time to time over the past 15 years(17-19) show that urine tends, irrespective ofstarting pH, to approach pH 6.9 at 250 C (or 6.6at 370 C); the phenomenon has not beenexplained.

Effect of urinary flow rate on urinary NH3 con-

centration

The concentration of the free base NH3 invoided urine at 370 C has been calculated with theaid of the Henderson-Hasselbalch equation fromconcentration of total ammonia and pH at 370 C,deriving pK'a from pKa, 8.890 at 370 C (20), byapplying the theoretical correction term for ionicstrength (21) as confirmed experimentally byBank and Schwartz (22). When the flow rateof urine nearly free of bicarbonate rises to 2 mlper minute, [NH31 appears to approach a low,limiting value and is little affected by further rise

40Urinary

[NH 30

p Eq.ml.

20x

IO-3

-

1o0-

30FUrinary

ENQ,js Eq.

ml.

x

20 F

10p-

0

0 00

0 0

14 1612

1695

40~


Subject H (*26)pH range 474-5.57

Q during Mannitol infusion(corrected for mean rate of decrease with time)

4.0+

3.0+

2.0+ ***

U * *4M **

*I U

* }* U

1.0+

2 4 6 8 10 12 14 16 16 20 22 24 26 28 30 32Urine Flow ml./min.

Subject R (*48)pH range 6.85 -7.24

© during monnitol infusion

40k

30 I-

10k

0

00

0

0

0

0

00

I I I I I I

1 2 3 4 5 6

0I*0I I I I a I I

8 9 10 11 12 13 14 15 1 17 1s 19 20 21

Urine Flow Rate mi./min.

FIG. 8. CALCULATED FREE-BASE [NHJ] IN VOIDED URINE. Mannitol was infused intrave-nously after a second peak of water diuresis in order to push urinary flow rate well abovethe physiologic ceiling. Subject H was pretreated with NHC1. During the 6-hour ex-

periment (no. 26), a small decrease of [NH3] occurred as a function of time, a decrease ap-parent in later low-flow as well as high-flow values. It was thought to reflect prior stimula-tion of renal deaminating activity, and has been corrected for.

in flow rate; two experiments representative ofseven such observations are depicted in Figure 6.

In urine containing more bicarbonate, [NH3]is higher, and the rise occurring at low flows isgreater and begins at higher flow rates than withmore acid urines; still, there appears to be littlechange of [NH.] as flow rate rises above 4 to 7

ml per minute, depending upon urinary pH.Figure 7 shows two experiments representativeof seven made at higher urinary pH.

In two of the experiments with acid urine, afterattainment of maximal water diuresis, mannitolwas infused intravenously in order to producehigher flows by osmotic diuresis. Little effect on

1696

r4)

0x

LU

FnIz0)0co

IL

Urinary[N H

s Eq.ml.

x

10-3© 0

1 1 I a


urinary [NH3] was observed in either case asflow rose above the physiologic ceiling, to over30 ml per minute in one experiment (no. 26, Fig-ure 8). In another experiment (no. 48, Figure8), done after the 45 experiments analyzed hereand similar to the former experiment except thaturinary pH was higher, osmotic diuresis againhad no apparent effect on urinary [NH3].

DISCUSSION

Effect of urinary flow rate on ammonia excretion

The data show that in normal man increasingurine flow rate by water-drinking increases uri-nary ammonia excretion, which confirms the mostsystematic studies previously reported of the ef-fect of water diuresis on ammonia excretion in un-prepared, healthy, fasting humans, those made 40years ago by Hubbard and Munford (23, 24).Nevertheless, the uniform effect of flow on am-monia excretion, irrespective of starting urinarypH, is not at present generally recognized; pos-sible reasons for seeming discrepancies betweenour findings and those of more recent investigatorswill be considered.

Prior reports in man. The most extensive ofmore recent relevant studies in man is that ofNutbourne and de Wardener (19), who concludedthat water diuresis does not increase ammoniaexcretion. As will be noted, the rate of H -secretion influences urinary ammonia excretionand to a much greater extent than flow rate. Ifammonia excretion is studied before and after in-duction of a single diuretic peak under experi-mental conditions that result simultaneously in asteady decrease of renal tubular H+-secretion, theeffect of flow on ammonia excretion is likely tobe obscured (although its effect on H+-excretionwill be overestimated). Such was the case inthe experiments of Nutbourne and de Wardener,in which renal tubular H+-secretion must havebeen falling from two causes as urinary flow in-creased. One cause was doubtless the feeding, 1hour before commencing observations, of a meal,which, because it stimulates gastric secretion ofH+, must cause temporary inhibition of renal tu-bular H+-secretion to a variable degree in differentsubjects. Another cause was presumably relatedto the 6-day premedication with NH4C1 up to 1hour before the experiments; its withdrawal

should occasion a progressive decline of total Heclaiming renal excretion. Data for a representa-tive experiment of Nutbourne and de Wardenershow that a decrease of tubular H+-secretion wasindeed occurring during the course of the experi-ment; they show a decline amounting to fully 30,uEq per minute in the rate of elimination of H+(as NH4+ and residual titratable acid) over the7 hours of observation of rising and falling flow.Similar declines occurred in the series of experi-ments as a whole. The same data show, nev-ertheless, that urinary flow increased on bothcontrol and experimental days and that the in-crease was attended both times by increasing uri-nary ammonia excretion during the period ofrising flow, provided data are corrected for fall-ing tubular H+-secretion.

MacKnight, MacKnight, and Robinson (25)found urinary ammonia excretion consistently in-creased during water diuresis in alkaline urine,but were unable to demonstrate any effect of flowin acid urine except when flow fell below 1 mlper minute. Data summarizing individual experi-ments with acid urine are confined to one graphderived from an unprepared subject and one froma subject premedicated with NH4C1. In thefirst experiment, there is a continuous and unex-plained fall of urinary pH, amounting to 2 pH Uthroughout the experiment; in the other, am-monia excretion appears to rise and fall with uri-nary flow. These investigators divided their dataon more acid urines into two groups according topH range and showed a single mean plot foreach group consisting of pooled data for four orfive experiments, each averaging about five indi-vidual urine collections. It seems not unlikelythat the considerable variation of these mass plotsmay be ascribed to pooling of data for urine ofvarying pH, and this variation may have obscuredan actual relation between flow and ammoniaexcretion.

The data reported by Richterich (26) are stillscantier; this author reports that the rate of am-monia excretion is affected by urinary flow, butbelieves that in acid urine this effect occurs onlyin the range of low flow. Supporting data, how-ever, are confined to part of one experiment.In the reports of Eggleton (27), Munck and Las-sen (28), and Ryberg (29), the effect of waterdiuresis on ammonia excretion was of secondary

1697


concern; in general, a relation to flow is discerni-ble, but the data do not permit unequivocal inter-pretation.

Reports in other species. Recourse may ofcourse be had to species differences to accountfor discrepancies with other data, but the publishedevidence for water diuresis exerting little or noeffect on the rate of ammonia excretion in acidurine is not really convincing. Richterich (26)interpreted his data to mean that herbivores (rab-bits and guinea pigs) differ markedly from manin their relation of urinary ammonia excretion topH, and also that the effect of flow rate on am-monia excretion is slight in acid urine. Never-theless, he states (26) that in the guinea pig''urine flow had a limiting effect at any urinarypH investigated." Orloff and Berliner (30)claimed that variations in the flow rate of acidurine in dogs (pH 6.2 or less) exerted no influ-ence on the rate of urinary ammonia excretion "ir-respective of the means of inducing diuresis," buttheir data did not include an examination of theeffect of water diuresis on ammonia excretions

Interpretations of the present report. A. Uri-nary flow rate as a determinant of urinary am-monia excretion-NH3 diffusion hypothesis.Changes in the rate of osmotic water reabsorptionappear unlikely to entail directly any majorchange in the rate of "active transport" of ionsor other solutes into or out of the nephron; in-deed, the current view that urine is concentratedby reabsorption of "solute-free water" reflectsthe fact that no considerable change is generallyrecognized in the rate of excretion of urinarysolutes, with the exception of urea, as urinary waterexcretion varies physiologically (1, 4). The firstsignificance, then, of our finding, in 45 successiveexperiments, that urinary ammonia excretionvaries with urine flow in man is that ammoniatransport by diffusion is probably concerned.Evidently, as water is osmotically abstracted fromthe distal part of the nephron, either the soluteswill be carried out with the reabsorbed water, or

3 Orloff and Berliner (30) induced "water diuresis" inexperiments with acid urines by administration of a "3to 5 per cent glucose solution delivering 100 to 200 iLMper min. of sodium sulfate." In the case of alkalineurines (where an effect of diuresis on ammonia excre-tion was recognized), "the sustaining infusion containedsmall amounts of NaHCO,, Na2HPO4, or KHPO4."

else, being restrained, their diffusion pressuremust rise; in either case, any highly diffusiblesubstance would probably be reabsorbed passively.

Urinary ammonia is known to have arisen denovo in the kidney as a result of deamination,principally of glutamine and, to a lesser extent,of certain amino acids brought to it by thecirculation (31-33). The great diffusibility ofthe free base NH3-greater than that of urea andnearly equal to CO2 (34)-has already given riseto the diffusion hypothesis (35) of urinary am-monia excretion. According to this hypothesis,NH3 diffuses through the renal parenchyma andpenetrates into both the renal venous blood andinto the acidified contents of the fluid within thelumen of the distal portion of the nephron. Thestrongest evidence for the diffusion hypothesishas been the urinary pH-dependency of urinaryammonia excretion: the more acid the urine, thegreater the rate of urinary ammonia excretion(23, 24, 30, 36-40). More than 98%7 of the totalammonia present in fluid at any physiologic pHis in the form NH+; most of the NH3 diffusinginto the nephron must therefore be instantaneouslyconverted to NH4+, but the extent of this con-version will increase as urinary acidity increases,so that the total ammonia transported by the time[NH3] is about the same inside and outside thelumen will be directly proportional to [H+] ofluminal fluid. The bulk of the ammonia is as-sumed to be transported at a site in the nephronat or below that where luminal contents are acidi-fied; this process has been supposed to consist ofexchange of the H+ secreted for Na+ reabsorbed(41) and assigned by most investigators to thedistal tubule (42-47). That certain organic weakbases introduced into the body are eliminated inurine at rates varying directly with urinary [H+]supports the diffusion hypothesis of urinary am-monia excretion (30, 35).

B. Further support for the diffusion hypothesis.The equivalence between increased ammonia ex-cretion and decreased titratable acid excretionthat is observed uniformly (Table II) as urinaryflow rate rises to its ceiling strongly supports thediffusion hypothesis. Since 99% or more of theammonia present in any fluid of pH less than 7 isin the form of NH.+, it follows that as NH3 dif-fuses into the lumen, it will be converted almostquantitatively to NH4+ by combining with H+

1698


according to the reaction: H+ + NH., diffusing in> NH4+. Where the bicarbonate content of urine

is low enough to be neglected,4 change of the rateof H+-excretion with changing urinary flow(NH4+ taken not to be a form of H+) can bemeasured as titratable acid, and equivalence be-tween increasing total ammonia excretion anddecreasing titratable acid excretion should beseen as urine flow rises, provided transport ofall the ammonia into the nephron is occurring bymeans of diffusion of NH3. If ammonia weretransported by active secretion of NH4+, as hasalso been suggested (48, 49), no such equivalencewould be expected, nor would there be evidentreason for the enhancing effects of either in-creased acidity or increased flow rate of urine uponurinary ammonia excretion.

Kinetic aspects of diffusion of NH3 into thenephron

NH3 might diffuse from the medullary inter-stitium into the nephron's lumen until the con-centrations inside and outside approach equi-librium; conversely, accumulation of ammonia inthe lumen might be limited because the rate offlux of NH3 from the sites of its production inthe renal parenchyma to the luminal fluid is in-sufficient to allow diffusion to proceed to equi-librium in the time required for luminal fluid totraverse the distal nephron segment. It has alsobeen suggested (30) that accumulation of am-monia in luminal fluid might be limited by therate of ammonia production when urinary pH is6.2 or less.

From the parallelism between the rate of excre-tion of urinary ammonia and the flow of urineat higher pH (> 6.2), recent proponents of thediffusion hypothesis have assumed (26, 30, 35)

4 All urine contains bicarbonate, but the amount issmall in urine of pH 6.0 and below, and rises ever morerapidly with rising urinary pH. Below what pH urinaryHC3O is negligible depends on the issue under considera-tion. For demonstrating equivalence of change of theexcretion rates of ammonia and titratable acid, as in Ta-ble II, urine up to about pH 6.3 at maximal flow can beconsidered to contain negligible HCO3-; for qualitativerise of pH with diuresis, experiments will serve in whichthe pH of high-flow urines is 6.5 or less at 370 C. Forexact calculations of the nephron's "H+ balance" withvarying flow, HC03- is negligible only when urinary pHat maximal flow is below 6.0.

that under these circumstances equilibrium con-ditions prevail across the nephron wall in re-gard to NH3. The assumption is doubtless cor-rect, although, as will be shown, the conclusionneed not follow from the premise. If equilibriumconditions are assumed, it follows that water reab-sorption distal to the locus of urine acidificationby H+-secretion would cause NH3 to diffuseback out of the nephron's lumen unless therewere a simultaneous, comparable rise of inter-stitial [NH3] radially toward the papilla, orthe nephron-segment distal to the site of NH3equilibration became relatively impermeable toNH3. Evidence against the former assumptionwill be presented.

Diminished permeability of the terminal neph-ron-segment to NH3? This assumption wasadopted by Milne, Scribner, and Crawford (35)in their mathematical treatment of the diffusionhypothesis, principally on the basis of data re-ported by others as showing that the flow rate ofacid urine has no effect on ammonia excretionrate. Our data confirm prior evidence (23, 24)that this is untrue, at least in man. The assump-tion that the terminal nephron-segment is poorlypermeable to NH3 is also incompatible with thenear-constancy of urinary [NH3] at all flowrates studied greater than 2 to 7 ml per minute(Figures 6-8), from which it also follows thaturinary [NH3] reflects interstitial [NH3] in theregion of the terminal nephron-segment, unlessurinary [NH3] changes beyond the papilla.

Limitation on accumulation of NH3 in the lu-men? The idea that accumulation of ammonia inthe nephron is limited during elaboration of urineof pH less than 6.2 by the rate of renal ammoniaproduction was also based on the hypothesis thatwater diuresis will not increase ammonia excre-tion under such conditions (30). There wasnever any sound experimental basis for this hy-pothesis, which would be very hard to reconcilewith the large increase of urinary ammonia ex-cretion resulting promptly when pH falls wellbelow pH 6.2.

Limitation of diffusion? Accumulation of am-monia in the lumen might be restricted, however,if the rate of flux of NH3 into the lumen were tooslow to permit establishment of equilibrium con-ditions within the time available for luminal fluidto pass through the distal nephron-segment.

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There would then exist an appreciable NH3concentration gradient between the renal paren-chymia and the luminal fluid. The parenchymal[NH3] would then be higher than under equi-librium conditions, and this would favor removalof ammonia through diffusion of NH3 into renaleffluent blood, thus assisting the renal veins intheir competition with the urine for removal ofthe ammonia produced in the kidney.

Limitation of the rate of diffusion of NH3 intothe nephron sufficient to prevent attainment ofequilibrium must become more and more con-spicuous as the quantity of ammonia being trans-ported into the nephron's lumen per minute rises.No significant limitation of transport of availableNH3 exists unless [NH3] inside and outside thelumen are not close to equilibrium by the timeluminal fluid has reached the terminal nephron-segment. Any NH3 concentration gradient de-veloping from renal parenchyma to lumen mustbe maximal during excretion of very acid urine,and limitation of diffusion could not be said to bevery important for renal ammonia excretion un-less under these conditions of maximal am-monia transport, [NH3] of the interstitial waterwere well above that of the urine at the papilla.

Diuresis could increase ammonia excretion un-der such conditions by increasing the volume ofluminal fluid per minute into which diffusion canoccur; for practical purposes, this volume is givenby the urinary flow rate. Since according toFick's law of diffusion the rate of flux of a dif-fusing solute is proportional to the concentrationdifference across the area of diffusion, one wouldexpect ammonia transport to be increased to theextent that the lowering of luminal [NH3] dur-ing diuresis increases the integrated NH3 con-centration difference across the nephron's wall.The lowering of urinary [NH3] by diuresis,however, is slight at most in urine of any pH.The largest proportional decrease of urinary[NH3] that might be concealed in the scatter ofthe points as the flow rate of acid urine rises pro-gressively above 2 to 4 ml per minute (Figures6-8) could represent an important increase in theconcentration difference across the luminal wallonly if urinary [NH3] were already very closeto interstitial [NH3] . This would mean, how,-

5 The following sample calculations clarify this point.Suppose, interstitial [NH3] to be 1.5 /uEq per L and

ever, that limitation of diffusion was of little orno significance under the conditions where itwould have to be most conspicuous. The scanteffect of raising urinary flow rate above 2 to 4ml per minute upon urinary [NH3] thereforeargues strongly against any important limitationon diffusion of NH3 from its sources to a con-centration inside the lumen approximating thatoutside it. Equilibrium conditions must certainlyprevail at high rates of urine flow, at least duringelaboration of urine of intermediate to maximalpH; the argument for back-diffusion of NH3 atlower flows becomes increasingly compelling asthe quantity of NH3 that must be transportedover the same gradient into the lumen diminishessharply with rising urinary pH.

Evidence for back-diffusion of NH3. A. To-pography of urine acidification by H+-secretionin relation to final water reabsorption. The argu-ment for diffusion of NH3 out of the terminalnephron-segment depends upon the validity of theview that urine acidification by H+-secretion iseffected mainly at a site proximal to completion ofurine concentration by water reabsorption. Thelatter process occurs throughout the collectingduct, at least during antidiuresis (4), whereasacidification by H+-secretion proper has been as-

luminal [NH3] to be 1.0 uEq per L at the papilla whenurine is maximally acid. Suppose [NH3] of the luminalfluid of the terminal nephron-segment drops 10% whenthe urinary flow rate of very acid urine rises from 2 to14 ml per minute; no more is compatible with the effecton urinary [NH3] of such an increase in flow rate asthe experimental series represented by Figures 6 and 8Ashow. It is easily calculated that there will result a maxi-mal increase of 20% over the initial value of the con-centration difference across the luminal wall. (Sinceurinary [NH3] scarcely changes, urinary NH3 excretionincreases almost 7-fold, but urinary ammonia excre-tion increases much less, Table I shows, owing to theassociated rise of urinary pH). Conversely, supposeinterstitial [NH3] under the same conditions to be fourtimes luminal [NH3], and suppose the same effect (a10%o decrease) of rising flow rate on luminal [NH3].The initial NH3 concentration difference across the lumi-nal wall is now 3 ,uEq per L, and it will increase 3.3%, to3.1 ,uEq per L). The evidence therefore indicates thatthe maximal decrease of urinary [NH3] possible to con-ceal in the scatter of urinary [NH3] of acid urine, as flowrises above 2 ml per minute (Figures 6 and 8A), couldreflect a sufficient increase of NH3 concentration dif-ference across the luminal wall to yield the observedincrease in urinary ammonia excretion only if equi-librium conditions had already been approximated.

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signed mainly to the distal tubules (42-44, 46,47).

Direct analysis has recently shown that luminalfluid is further acidified within the collecting ducts(45, 46, 48), but the process does not necessarilyconsist entirely or principally of H+-secretionproper. Ullrich and his colleagues found that H+and ammonia were added to, and Na+ reabsorbedfrom, the lumen of the nephron between the far-thest zone to which the catheter could be passedand a site in the collecting duct located closer tothe papilla. Urine collected through the bladderfrom the uncatheterized kidney, however, wasregularly more acid (in 23 of 24 experiments)than fluid simultaneously collected through acatheter with its tip placed at locations nearerthe papilla (45), whereas ammonia concentra-tion at these latter sites was higher than that ofbladder urine in 2 out of 8 experiments and ofthe same order of magnitude in 3 to 4 of the re-maining 6 experiments (48). Water was beingreabsorbed from the collecting duct, although theexperiments were made under diuretic conditions(48). H -generation resulting from diffusion ofNH3 out of the luminal fluid most directly ex-plains about half of these data, although the au-thors did not so conclude; the other half wouldbe expected if both H+-generation owing to back-diffusion of NH3 and H+-secretion occur distallyto the region sampled.

These experiments were performed on thegolden hamster, whose exceptionally well-devel-oped collecting ducts and single papilla permitpassage of catheters deep into the inner medulla.In this species, and perhaps in others withanatomically similar kidneys, the prominent col-lecting duct may take over a considerable por-tion of H+-secretion reserved to the distal tubulein other species. The experiments described wereconducted under conditions of forced diuresis andusually of acidosis as well, so reabsorption of NH3might possibly or even probably begin consider-ably higher in the collecting duct during antidiu-resis in hamsters. The postulated generation ofH+ through back-diffusion of NH3 can fully ac-count for the apparent discrepancies in the locusof H+-secretion between the reports of Ullrichand his colleagues (45, 48) and those of investi-gators working with other mammals and withamphibia (42-44, 47).

Gottschalk, Lassiter, and lIylle (46) have re-cently reported that the pH of luminal fluid ob-tained by micropuncture from nephrons of ratsdeclines in the collecting duct, and that the fallis much greater during antidiuresis. Taken to-gether with the evidence presented here that vari-ation of urinary flow rate in man affects neithertotal H+-elimination nor H+-secretion proper, thelatter observation suggests that acidification ofcollecting duct fluid is due to H+-generation re-sulting from diffusion of NH3 out of the collectingduct rather than to H+-secretion proper.

B. Summiarized evidence for back-diffusion ofNH3. Wehave rejected the hypothesis of limita-tion of diffusion in favor of the view immediatelysuggested by the near-constancy of values for urine[NH3] above flow rates of 2 to 7 ml per minute,namely, that NH3 diffuses to equilibrium in thenephron's lumen at and below the site of H+-se-cretion proper, and that equilibrium conditionsare thereafter maintained as the luminal fluidproceeds to the papilla. The constancy of thesum of titratable acid and ammonia excretion innearly bicarbonate-free urine will also be main-tained as NH3 diffuses back: NH4+ -> H4 + NH3(diffuses off).

Water could be reabsorbed from the NH3-permeable terminal nephron-segment withoutcausing back-diffusion of NH3, provided the in-terstitial [NH3] rose toward the papilla so as toequal or exceed the rise in luminal [NH3] thatwould be effected by reabsorption of truly "solute-free" water in the amount reabsorbed from thatsegment. Our data cannot exclude this possi-bility, nor can they explain the progressive rise ofurinary [NH3] occurring, more sharply in al-kaline urine, as urinary flow falls below a criticalvalue, which is influenced by urinary pH (Figures6-8). Quantitative kinetic analysis of these andother data (50) is required to explain why [NH3]rises conspicuously in low-flow urine.

Additional evidence indicates back-diffusion ofNH3 during the excretion of nearly bicarbonate-free urine, and more of it as urine flow falls.By the equilibrium back-diffusion model, theremust be a point in the distal nephron beyondwhich H+ simultaneously appears in luminal fluidas an equivalent amount of ammonia disappearsfrom it; also, back-diffusion of NH3 in proportionto water reabsorption should result in increased

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acidity of collecting duct fluid during antidiuresis.We have cited evidence indicating that boththese phenomena occur (45, 46, 48). This evi-dence is difficult to reconcile either with limi-tation of diffusion or with a rise toward the papillaof interstitial [NH3] sufficient to prevent back-diffusion, since in both cases He should be con-sumed instead of generated in the collectingduct as NH3 continued to diffuse into the luminalfluid.

Relation between change of residual titratable acidand change of urinary pH with changing uri-nary flow rate

Nutbourne (51) has drawn attention to the na-ture and magnitude of the rise of urinary pH thatis ascribable to the purely physicochemical ef-fect of dilution; this phenomenon is caused bychange in the activity coefficient of phosphatic andother urinary buffer ions as predicted by thelimiting equation of Debye and Hfickel. To as-certain the contribution of this physicochemicaleffect to the rise of urinary pH produced by waterdiuresis, we diluted low-flow urine samples, fromfive experiments with very acid urine, immediatelyafter voiding with water in vitro so as to simulatethe difference in the volume voided per minuteover the range of flow observed in the correspond-ing experiments. The resulting increase in invitro pH was calculated for each experiment frompooled, multiple, linear regressions of pH on thelogarithm of sample dilution; it averaged 0.095pH U (range, 0.072 to 0.117). The in vitrochange averaged 14%o and never exceeded 18%of the difference in pH between low-flow andhigh-flow urine.

SUMMARY

1. Urinary excretion of total ammonia regularlyrises, irrespective of initial urinary pH, whenurine flow of healthy men is caused to rise fromaround 1 ml per minute to the physiologic ceilingby water-drinking. The absolute increase during,diuresis is considerably greater in acid than inalkaline urine; the percentile increase is greatestfor the highest urinary pH range (neutral to al-kaline), decreasing progressively as urinary pHdeclines.

2. When urinary pH is less than 6.4 dur-ing water diuresis, the increase of ammonia ex-

cretion is accompanied by a decrease, approxi-mately chemically equivalent, in the rate of ex-cretion of residual titratable acid. The equiva-lence supports the view that the rate of elimi-nation of ammonia in the urine is governed bythe amount of net diffusion of NH3 out of therenal parenchyma into the fluid passing throughthe lumen of the distal portion of the nephron,since enhanced net diffusion of NH3 into thelumen as urinary flow increases will diminish H+-excretion by converting H+ to NH4+ while simul-taneously increasing total ammonia excretion.

3. Urinary pH varies with urinary flow rate,tending always to approach 6.6 at 370 C as flowrises. When the pH of low-flow urine is 6.0 orless, it increases about 0.5 to 1.0 pH U as flow raterises from 1 ml per minute to its ceiling. Lessthan 20% of the change can be ascribed to thephysicochemical effect of dilution; the remainderappears to be due entirely to increased net diffu-sion of NH3 into luminal fluid.

4. Urinary concentration of free-base NH3 islittle affected when the flow rate of neutral toalkaline urine rises above about 4 to 7 ml perminute, or the flow rate of acid urine above about2 ml per minute, to and well beyond the physio-logic ceiling of water diuresis. The findings areincompatible with impermeability to NH3 of theterminal nephron-segment, where water reabsorp-tion is completed, and indicate equilibration un-der these conditions of [NH3] of the luminal fluidwith that of the adjacent interstitial water. Semi-quantitative treatment of the data provides evi-dence, reinforced by more direct published data,indicating that NH3, after diffusing to equilibriumin the acidified luminal fluid of the distal nephron-segment, subsequently diffuses out of the terminalnephron-segment as a result of and in proportionto osmotic reabsorption of "solute-free water."

ACKNOWLEDGMENT

Weare indebted to Dr. Richard Singer for his help-ful advice.

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