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The renal mechanism for urate homeostasis in normal man
The Renal
Mechanism
for Urate
in Normal THOMAS H. STEELE,
M.D.~
Homeostasis
Man*
and RICHARD
E.
RIESELBACH,
M.D.
Madison, Wisconsin In thirty-two studies on ten normal subjects, renal urate secretion and reabsorption have been estimated by noting the decrement in UVurate: Clnulin produced by a maximally effective dose of pyrazinamide (TS,,), a drug which markedly although probably not completely inhibits tubular urate secretion. In each subject studies were performed in the control state, after decreasing plasma urate with allopurinol, and following elevation of plasma urate levels by RNA loading. Urate reabsorption remained at an average of 98 per cent of filtered load at all plasma levels, thereby indicating progressive augmentation of reabsorptive transport velocity with increasing filtered loads. Also, tubular secretion per nephron was a direct function of plasma urate concentration. Although plasma urate levels ranged as high as 12.8 mg. per 100 ml., at no time was there evidence for attainment of a secretory or reabsorptive tubular transport maximum. A provisional normal standard was set forth, relating TS,, to plasma urate concentration through a regression treatment with appropriate confidence limits. These data suggest that normal man displays an augmented rate of bidirectional urate transport with increasing substrate availability. Due to the nature of this bi-
directional transport system, an increase in secretory rate serves to increase net urate excretion and thereby provides the homeostatic mechanism which tends to minimize the hyperuricemic response to an increase in uric acid synthesis. The pyrazinamide suppression technic appears to provide a means of assessing the integrity of this homeostatic mechanism within the residual nephrons of the chronically diseased kidney or in patients who have gout. cult in that the renal excretion of urate is governed via a bidirectional transport system. There is now convincing evidence that filtered urate is virtually completely reabsorbed; that urate which appears in the final urine apparently gains access predominately through active secretion [3,4]. Studies performed to date suggest that at least a twofold increase in plasma urate concentration would be necessary to exceed the maximum rate of tubular reabsorption (T,) for urate [5]. Therefore, an increase in filtered load of urate, secondary to an increased plasma urate concentration, would not serve as a regulatory mechanism until marked hyperuricemia supervened unless the reabsorptive mechanism exhibited a marked degree of splay.
normal man excessive ingestion of uric acid precursors usually results in a significant increase in the rate of urinary urate excretion in association with only a modest increase in the plasma urate concentration [I]. Similarly, in some myeloproliferative disorders minimal elevations in plasma urate concentration may be accompanied by marked increases in the urinary excretion of urate [2]. Thus the human kidney may significantly increase its rate of urate excretion in the presence of even a modest degree of hyperuricemia, thereby serving to minimize the hyperuricemic response to ex*;enously or endogenously derived urate loads. Characterization of the mechanism underlying this augmentation of excretory rate is diffi-
I
N
* From the Department of Internal Medicine, The University of This study was supported by U. S. Public Health Service Research and The Wisconsin Alumni Research Foundation. Requests for Rieselbach, M.D., Department of Medicine, University Hospitals, 53706. Manuscript received December 16, 1966. t Postdoctoral Research Fellow, U. S. Public Health Service. Center, U.S. Public Health Service Hospital, Baltimore, Maryland.
868
Wisconsin Medical School, Madison, Wisconsin. Grants AM 09943-01, AM 05630, FR 5435-05 reprints should be addressed to Richard E. 1300 University Avenue, Madison, Wisconsin Present
AMERICAN
address:
Baltimore
JOURNAL
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MEDlClNE
Renal
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It appears more reasonable to examine the secretory component of the bidirectional transport system for urate. There is evidence to suggest that urate may share the same secretory mechanism as other organic acids such as paraaminohippurate [6]. Therefore, it is reasonable to postulate that, just as for para-aminohippurate, the velocity of mate transport from renal peritubular fluid to tubular lumen should increase as substrate availability increases, until a maximum is reached where intrinsic tubular factors might become rate limiting. In the present studies an attempt has been made to examine the rate of urate secretion per nephron in ten normal control subjects by observing the change in the urinary excretion of urate per unit of glomerular filtration following suppression of urate secretion by the administration of pyrazinamide. By conducting a minimum of three separate studies on each subject at different plasma urate levels (attained by pharmacologic manipulation), data were obtained which confirm the presence of a sensitive regulatory mechanism for urate within the kidney. This system contributes to urate homeostasis through an alteration of secretion rate in response to changes in the plasma urate level. An attempt has been made to quantify this relationship so that the appropriate secretory response to a given plasma urate level may be defined within predicted limits in normal man. MATERIALS
AND METHODS
A total of thirty-two studies were performed in ten normal volunteer subjects. This group was composed of eight male physicians and one male and one female patient from the general medical service. None of the subjects had evidence of renal disease on the basis of history, physical examination or laboratory findings. All subjects were Caucasian except for V.W., who was of Oriental extraction. Each subject underwent a minimum of three studies. Each study consisted of two phases; the first phase involved at least three clearance periods of fifteen to thirty minutes in duration. Following this 3 gm. of pyrazinamide was given orally. After an interval of approximately one hour, the first of three additional clearance periods of similar duration was initiated. The majority of subjects exhibited maximum suppression of urinary urate excretion in either the second or third period after the administration of pyrazinamide. During the three clearance periods following the dose of pyrazinamide, however, the rate of urinary urate excretion did not vary by more than 10 per cent from the average of the baseline periods. Therefore, it seemed likely that maximal suppression VOL.
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1967
Homeostasis-Steele,
Rieselbach
was being observed within the time interval employed, and that carrying out the clearance procedure for a longer time would be of little value. Standard clearance technics were employed [7] ; urine flow of 7 to 15 ml. per minute was induced by aggressive oral hydration so that all clearance studies could be carried out without catheterization. Each subject was studied at a minimum of three different plasma urate levels. The first study was performed at that subject’s endogenous plasma urate level. Then allopurinol* (4-hydroxpyrazolopyrimidine) was administered for seven days at a dose of 600 to 800 mg. per day in order to reduce the plasma urate concentration by decreasing the rate of urate synthesis through xanthine oxidase inhibition. The second study was carried out after allopurinol had been discontinued for twenty-four hours in order to allow time for excretion of the drug, its primary metabolite, alloxanthine (4,6-dihydroxypyrazolopyrimidine), and the oxypurine byproducts of enzyme inhibition. The third study was performed a minimum of one week following the second study. Prior to this study 4 gm. of yeast ribonucleic acid (RNA) was administered daily for four days. In two subjects a fourth study was carried out following further RNA or intravenous uric acid loading. Glomerular filtration rate was determined by the clearance of inulin; inulin analyses were carried out according to the method of Roe et al. [a]. Uric acid determinations were performed by the enzymatic apectrophotometric method of Liddle et al. 191. RESULTS
Estimation of Tubular Secretion of Urate ( TS,,) by the Pyrazinamide Suppression Technic. In every study the administration of pyrazinamide resulted in a sharp decrease in urinary urate excretion. Plasma urate levels were essentially unchanged from the first to the second phase of each study. Because of the desirability of expressing excretion relative to a given population of nephrons, urate excretion values are factored by the inulin clearance, thus giving an estimate proportional to urate excretion per nephron. t In the representative study illustrated in Table I, inulin clearance and plasma urate level remained quite constant. The mean excretion of urate during the control periods was 4.23 pg. per minute per glomerular filtration rate (or pg. per ml.). Fifty-eight minutes following the oral administration of 3 gm. of pyrazinamide, excretion * Zyloprim@, Burroughs Wellcome 56-l 58.
t Hereafter, for the sake of brevity, the term excretion per nephron wiil be employed interchangeably with excretion per milliliter of glomerular filtrate. However, it is to be understood that the two are not synonymous, but that the former is merely proportional to the latter, and is of much smaller magnitude in absolute terms.
870
Renal
Mechanism
for Urate
Homeostasis-Steele,
TABLE PROTOCOL
I
OF A REPRESENTATIVE SUBJECT
Rieselbach
STUDY
W.S.
C
Cinulin (ml./min.)
0 43 61 81 101 Mean values
Begin clearance
159
TS,,
(mg.Ikf?ml.)
C (ml.~rZn.)
Cinulin
FE,,,, (estimate
-
administration;
1.0 1.3 1.7 1.34
4.2 4.4 4.2 4.23
7.0 7.2 7.1 7.11 empty bladder
0.51 0.66 0.80 0.657
0.90 1.1 1.4 1.15
uvurste decrease in Cinulin induced by pyrazinamide
= maximum = (4.23)
periods after pyrazinamide 5.7 5.8 5.6 5.71
116 111 122 116
Cinulin (rg/mi.)
(%)
Inulin prime and sustaining infusion Begin clearance periods; empty bladder 8.7 5.9 124 9.0 6.1 126 9.1 5.8 128 8.96 5.94 126 Pyrazinamide, 3 gm. given orally
179 195 211 Mean values
NOTE:
UVurate __
2%x~oo
Time (min.)
(0.51)
= maximally of fraction
= 3.72 pg./min./Ci,,ii, (estimate of urate secretion C depressed Cir:,tL ~ after pyrazinamide = 0.90 per cent.
per nephron).
of filtered urate escaping reabsorption).
per nephron (UV,,,,, :Cinulin) had decreased to 0.51 pg. per ml. Although this is the minimum level of excretion observed under secretory inhibition, the values during consecutive time intervals are similar. In order to assess urate secretion as quantitatively as possible, the minimum value of excretion (at maximal drug effect) was compared to the control mean excretion value. In this particular study UVurate :Cinulin decreased 88 per cent. If this decrease in urate excretion occurred as a result of secretory inhibition, then tubular secretion must account for at least 88 per cent of urinary urate excretion in this subject at a plasma urate concentration of 5.9 mg. per 100 ml. Tubular secretion of urate per nephron (TS,,) is then calculated as the mean initial excretion per nephron, 4.2 pg. per ml., minus excretion per nephron at maximal pyrazinamide effect, 0.51 pg. per ml. The result 3.7 pg. per ml., is regarded as an estimate of the secretory rate per unit of glomerular filtration rate, and hence is proportional to the secretory rate per nephron. In addition to control studies, all subjects were restudied on different occasions at a minimum of two other plasma urate concentrations. Plasma urate was decreased through xanthine oxidase inhibition by allopurinol, or increased through the administration of yeast RNA.
The results of all studies are given in Table II. In every subject a decrease in plasma urate level resulted in both decreased total urate excretion and urate secretion per nephron. Likewise, elevation of the plasma urate concentration was accompanied by elevated excretion and secretion rates. Response of Tubular Reabsorptive Rate to Change in Filtered Load of Gate. A summary of all the data (Table III) indicates that the percentage decrease in urate excretion following the administration of pyrazinamide was essentially the same, irrespective of the plasma urate level. Thus, at all three plasma concentrations secretion appeared to constitute a relatively stable percentage of excretion, averaging approximately 80 per cent. Since it is reasonable to consider that the decrement in urate excretion produced by pyrazinamide occurs solely through inhibition of secretion, any remaining urate excretion during maximal action of pyrazinamide reflects the amount of filtered urate which has escaped reabsorption during passage through the nephron, if suppression of tubular secretion of urate by pyrazinamide is assumed to be complete. Hence, fractional urate excretion (FE,,.&,) = nonreabsorbed filtered urate : total filtered Simiurate = Curate:Cinuiin after pyrazinamide. larly, an estimate of the fraction of filtered urate AMERICAN
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Homeostasis-Steele,
reabsorbed would be 11 - (Curute:Cinur,nafter pyrazinamide)]. From Table III, it may be seen that the mean FE,,,,, approximates 2 per cent of filtered load at all plasma mate levels. Furthermore, in individual subjects (Table II) FEurat, is a relatively stable quantity irrespective of plasma urate concentration. Thus, urate reabsorption was proportionally augmented with increasing filtered loads (Fig. 1) ; a reabsorptive T, was not reached with the plasma urate concentrations employed in these studies. Response of Urate Secretory Rate to Changes in Plasma Urate Concentration. As shown in Table increased or decreased appropriately II, TS,, with induced changes in plasma urate concentraTABLE COMPLETE
DATA
Rieselhach
871
tion in all patients studied. At a mean plasma urate of 5.35 mg. per 100 ml., mean TS,, was 4.01 pg. per ml. (Table III). Lowering of plasma urate in all subjects to a mean of 3.26 mg. per 100 ml. after allopurinol administration resulted in a decrease of mean TS,, to 1.93 pg. per ml. Elevation of plasma urate to a mean of 8.40 mg. per 100 ml. after RNA loading resulted in an augmentation of mean TS,, to 8.40 pg. per ml. Determinations of 4,6-dihydroxypyrazolopyrimidine levels were carried out in three subjects by Dr. Gertrude Elion of BurroughsWellcome Co., employing an isotope dilution method [lo]. These subjects had received 800 mg. of allopurinol per day for one week, and at II
ON TEN NORMAL
SUBJECTS
Secreted Urate Age (yr.) and Sex R.H.,
Cinulin
(ml./min./ 1.73 M2.)
32, M
(113)
T.S. 29, M
(119)
G.S., 41, F
(114)
V.W.,
(101)
32,M
W.S., 29, M
(110)
R.R.,
M
(106)
A.M., 28, M
(107)
A.K.,
30, M
(108)
F.G., 29, M
(96.9)
R.C.,
(117)
32,
26, M
C “r*ta
Plasma Urate
Patient,
Cinulin x 100
Study* Control HPP RNA (1) RNA (2) Control HPP RNA (1) RNA (2) Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA
(%) 5.8 3.8 9.0 12.8 5.8 4.3 7.9 12.2 3.8 2.5 5.3 4.3 2.8 6.3 5.9 3.4 9.6 4.8 3.1 9.1 4.8 3.7 7.4 6.1 2.8 7.0 6.4 3.3 7.1 5.6 2.9 7.1
518 215 920 1,458 598 299 695 1,853 560 402 808 670 339 1,279 470 222 942 355 202 1,402 350 272 679 583 261 1,068 539 269 1,021 769 218 906
7.0 4.8 10.3 10.6 8.8 5.4 7.5 14.0 14.5 14.1 11.9 16.7 11.3 19.4 7.1 5.8 9.3 6.6 6.5 14.4 8.0 7.4 7.1 8.6 8.3 15.3 9.0 8.1 15.2 11.8 6.7 10.6
* HPP = studies performed after one week of 4-hydroxypyrazolopyrimidine formed after four days of ribonucleic acid administration. VOL.
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1967
TS,, (pg./ml.) 2.7 1.3 7.8 9.8 4.3 1.9 5.0 15.5 2.9 1.3 3.8 6.1 3.0 11.7 3.7 1.8 8.2 2.5 1.7 10.3 3.6 2.4 4.7 4.1 2.1 9.3 4.9 2.3 8.7 5.2 1.5 6.2 administration;
(%)
Excreted Urate x 100 (70)
2.6 1.5 1.6 3.0 1.4 0.9 1.1 1.3 6.6 9.1 4.7 2.7 0.8 0.9 0.9 0.2 0.9 1.3 1.2 3.1 0.6 0.8 0.8 1.8 0.6 2.1 1.2 1.3 2.8 2.6 1.8 1.9
65.7 70.7 84.5 72.3 84.2 83.0 84.9 91.2 52.2 36.5 60.2 84.7 92.8 95.1 87.9 95.3 91.1 80.1 83.3 78.6 92.5 89.2 89.3 78.7 92.2 86.6 86.2 85.2 80.2 79.0 75.9 82.8
FEu,ate X 100
RNA
= studies per-
Renal Mechanism
872
for Urate
Rieselbach
Homeostasis-Steele,
TABLE III SUMMARY OF DATAIN TABLE II IIPP Data
Cinulin
(ml./min./l.73 Plasma urate (mg./lOO ml.) UV”&? (pg./min.) C (yy- x 100
Control
RNA
Mean
S.E.
Mean
S.E.
Mean
S.E.
111 .o
(2.9)
108.0
(3.9)
110.0
(3.3)
Ms.) 3.26 270.0
(0.17) (20.0)
5.35
(0.27)
8.40
(0.66)
541 .o
(41 .O)
1,087.O
(101 .O)
12.1
(1.05)
7.83
(0.91)
9.80
(1.08)
TS”,~
1.93
(0.17)
4.01
(0.37)
8.40
(0.96)
(P&/ml.) (FL.t.) X 100
1.83
(0.82)
2.15
(0.54)
2.01
(0.34)
,Il”IllJ
(%)
(%)
(Secreted
Urate)
(Excreted
Urate) >
(%)
x
80.4
100
(5.5)
79.1
(3.8)
83.1
NOTE: HPP = studies performed after one week of 4-hydroxypyrazolopyrimidine administration. performed after four days of ribonucleic acid administration. See text for definition of parameters.
the time of study, twenty-four hours after stopping administration of the drug, values ranged from 0.78 to 0.88 mg. per 100 ml. The plasma oxypurine level in one subject at this time, also determined by Dr. Elion, was 0.1 mg. per 100 ml. Although definitive studies in man have not been carried out as yet it is unlikely that these levels of drug metabolite and enzyme inhibition by-product, respectively, influenced urate secre120 100 60 60
FILTERED URATE
: G,,,,,
(JW/ml.)
FIG. 1. The proximity of the points to the d:agonal line of complete reabsorption at all plasma urate levels indicates virtually complete reabsorption of filtered urate (mean 98 per cent throughout the range of filtered load studied). TR,,,, = P,,.,. (I - Curate: Cinurinafter pyrazinamide administration), a minimal estimate of urate reabsorption per nephron.
(2.8)
RNA
= studies
tion or excretion to a significant degree. Thus, in each subject the only variable was the plasma urate concentration. Therefore, it is reasonable to ascribe the change in secretory rate to variations in this parameter. Predictability of Urate Secretion in Relation to Plasma Urate Concentration. One goal of these studies has been to derive a standard nomogram which would establish the normal range of urate secretion (TS,,) at a given plasma urate concentration. The data presented in Table II reveal that the secretory response to a given plasma urate level is not identical from one subject to another. Figure 2 (tok), in which TS,, is plotted as a function of plasma urate, graphically illustrates the wide range of secretory response encountered in five subjects. Two subjects (T.S. and R.H.) who had received additional RNA and sodium urate intravenously, respectively, exhibited acceleration and deceleration of TS,,, respectively, at plasma urate levels over 12 mg. per 100 ml. One subject (V.W.) never became hyperuricemic after RNA loading, presumably because of a brisk rate of urate secretion (TS,, 11.7 pg. per ml. at a plasma urate of 6.3 mg. per 100 ml.). Another (A.K.) exhibited brisk acceleration of TS,, as soon as the hyperuricemic state was attained. With one exception (V.W.), values in all subjects tended to cluster TS,, together during studies at normal and decreased plasma urate levels. AMERICAN
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Figure 2 (bottom) depicts urate to inulin clearance ratios in the same five subjects, expressed as functions of plasma urate concentration. The relationship between TS,, and C&,: C inulin is inconstant because of the artifact introduced into the latter by factoring excretion per nephron by plasma urate [Curate: Cinuiin = is uvurate : Cinuiin X 1 : PuratP]. Although TS,, a nearly linear function of plasma urate in R.C., C urate: C in” iin actually decreases upon attainment of hyperuricemia. Likewise, Curate: Cinuiin remains constant in R.H. as the uppermost value of plasma urate (12.8 mg. per 100 ml.) is approached. Thus, since parallelism between is not always mainC “rate: Cinuiin and TS,, tained, the former parameter appears to be poorly descriptive of urate secretion. In attempting to relate TS,, to plasma urate, separate linear regression treatments of studies at normal and elevated plasma urate levels
873
Rieselbach
Homeostasis-Steele,
(excluding G.S. and V.W., two subjects who did not achieve a plasma urate over 6.5 mg. per 100 ml. after RNA loading) revealed values of standard error of the estimate of 1.073 and 2.443, respectively. The variance of TS,, is significantly greater at hyperuricemic levels (F = 5.145; P < 0.01) than at lower plasma urate concentrations. Therefore, a linear regression treatment of TS,, and plasma urate, with common variance, is not justified. A log-log regression treatment of all data is depicted in Figure 3. The resulting nonlinear regression obtained is concave upward and has a gradual increase in variance as TS,, and P,, increase. In Figure 3 the 95 per cent confidence limits of the regression itself, and the 95 per cent prediction confidence limits for individual data, are depicted graphically. Although the prediction confidence limits could be narrowed somewhat by eliminating subject V.W. from the
PLASMA URATE (mg/l~mt.)
16
II
2
I,
4
I,
6
Ill
I,
8
10
12
PLASMA URATE (mq/lOOml.)
I
I 2
I
I 4
I
I
,
6
P~JYDM URATE
I 8
,
I IO
,
1
,
12
(mg./lOOmL)
FIG. 2. Top, TS,,
is plotted as a function of plasma mate concentration in five subjects. Note the wide range of secretory response observed at each level of plasma mate. Bottom, C”,., : Cinul in plotted as a function of plasma mate concentration for the same five subjects. Note the inconstant relationship of Curate :Cio”lin to the actual rate of mate secretion (TS,,). VOL.
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1967
Fig. 3. Log-log regression treatment of TS,, on plasma mate in thirty-two studies on ten normal subjects, replotted on a linear scale. The regression equation, TS,, = 0.413 (plasma mate)‘.%9 is indicated along with the correlation coefficient. The 9.5 per cent confidence limits of the regression (f0.0559 log units) are indicated by the darkly shaded area. The 95 per cent confidence limits for prediction of normalcy (f0.331 log units) are indicated by the lightly shaded area. The one point lying outside the 95 per cent prediction confidence limits is that of V.W. after RNA loading. Note that this subject did not achieve hyperuricemia because of a brisk secretory response. Variance of the secretory rate per nephron (TS,,) is significantly increased in hyperuricemia (see text).
874
Renal Mechanism
for Urate
regression, there is no definite justification
for removing these data from the study. Thus the TS,, of a subject would not be considered distinctly abnormal unless it happened to lie outside the 95 per cent prediction confidence interval of Figure 3. COMMENTS
A technic employing the administration of a large, single dose of pyrazinamide has been utilized as a means of quantitatively estimating the component of renal urate excretion which is derived from an active tubular secretary process. In addition, if complete filtration of urate at the glomerulus may be assumed, an estimate of urate reabsorption within the nephron was obtained. Because of the probability that complete inhibition of the secretory mechanism was not attained with this technic, the estimates obtained are those of minimal secretion and reabsorption at a given plasma urate level. Admittedly these data are based upon the assumption that the decrease in excretion of urate following the administration of pyrazinamide results from suppression of secretion, as suggested by stop-flow studies in the dog [II]. It is difficult to conceive that pyrazinamide could act by virtue of enhancing the rate of urate reabsorption, although there is no evidence in man to rule out this possibility unequivocally. The similar degree of apparent secretory suppression between different subjects and between individual studies in the same subject suggests that this technic is of adequate reproducibility. The dose of 3 gm. of pyrazinamide is more than employed in most previous studies in man [ 721; this dosage level was established in order to minimize variations in response which might occur due to different rates of drug absorption within individual patients. Although this technic is not completely quantitative, we believe that it represents a significant advance in assessing the renal mechanism for urate excretion. Most previous studies have attempted to evaluate the renal excretory capacity for urate by employing the parameters of UVurate:Ginuiin and Gurate:Ginuiin ]7,73,74]. Both parameters would appear to be nonspecific because they represent the resultant effect of two independent processes, as previously pointed out by Yti et al. [75]. The urate to inulin clearance ratio is particularly lacking in physiologic significance. Although this parameter gives the ratio of excreted to filtered urate, the pyrazinamide suppression data reveal that a maximum
Homeostasis-dteele,
Rieselbach
of only 2 per cent of filtered urate is excreted on the average. Thus, since the bulk of urinary urate is derived from tubular secretion, there appears to be no justification for factoring the amount excreted by the filtered load. This parameter might correlate reasonably well with TS,, in some patients, but the present data suggest that the correlation is quite unpredictable and is not sufficiently quantitative to serve as a means of comparing the renal response to hyperuricemia in any group of patients (Fig. 2.). The application of the technic described herein considerably increases the specificity of information which may be derived from studies of urate excretion, as may be further emphasized by the following example. It has already been noted that urate reabsorption is nearly complete within the normal nephron with mean values of FEurate of approximately 2 per cent at all plasma levels studied. Two subjects (G.S. and V.W.) had control urate to inulin clearance ratios of 14.5 and 16.7 per cent, respectively; on this basis each would formerly have been classified as having a “reabsorptive defect for urate.” However, the FEurate value in one (G.S.) was 6.6 per cent, whereas in the other (V.W.) it was only 2.7 per cent. The increased excretory rate of the latter subject was due to a markedly increased rate of secretion rather than a deficient rate of reabsorption. Thus, this technic allows estimation of secretion and reabsorption as independent events-each with definable normal limits. The formulation of TS,, involves the utilization of glomerular filtration rate as a basic index of nephron population. As long as the average glomerular filtration rate per nephron remains relatively constant, the total glomerular filtration rate will reflect the number of functioning nephrons. Therefore, the expression of urate corrects for losses in secretion “per nephron” the functioning nephron population incurred through disease, and thus provides a means of evaluating the performance of nephrons within the context of their functional mass at a given point in time. In addition, voiding errors become less critical because of the use of only urine and plasma concentrations in the calculations. It has been suggested previously by Wyngaarden [76] that renal urate secretion, expressed as a function of plasma urate level, may describe a sigmoidal pattern which initially accelerates with increasing plasma urate levels, and then reaches a plateau as a hypothetical tubular secretory maximum is approached. On the basis of our data we can only conclude that AMERICAN
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Renal
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such a secretory maximum must occur only at very high plasma urate levels. However, over the range of plasma urate concentrations employed in the present studies, the data might be considered to be consistent with the kinetics of an enzyme system displaying a sigmoidal velocitv-substrate relationship. ‘These data suggest that normal man is capable of augmenting both reabsorptive and secretory transport in the face of increased substrate availability. Theoretically, by employing sufficient urate loading and utilizing pyrazinamide a reabsorptive and secretory T, suppression, should be demonstrable. The marked augmentation of urate reabsorption with increased filtered loads serves to protect the nephron from an overwhelming load of urate in the presence of hyperuricemia. However, the secretory component of this bidirectional transport system appears simultaneously to attain an increased rate of activity. The net effect is a comparatively moderate increase in urate excretion. Nevertheless, the degree of hyperuricemia resulting from the accession of a given quantity of urate to the extracellular space is modified, and a reasonable degree of homeostasis is maintained. As an extension of the foregoing thesis the question arises as to the integrity of these transport systems in disease. The experimental technic described should provide a means of determining whether these transport systems remain intact within the residual nephrons of the chronically diseased kidney, thereby enabling these units to respond appropriately to a given degree of hyperuricemia and effectively contribute to homeostasis. Also, this technic should provide a precise means of characterizing the renal mechanism for urate excretion in gout. If the etiology of gout in some patients is clearly associated with a deficient rate of urate secretion, it is reasonable to expect that TS,, in these patients at hyperuricemic and pharmacologically induced normouricemic levels should distinctly differ from that of normal subjects. The great variability of TS,, at elevated plasma urate levels in normal subjects emphasizes the need for well defined normal limits of excretory rate at a given plasma urate level if the excretory mechanism in patients with gout is to be compared to that of normal subjects. Acknowledgment: We are indebted to Dr. Gertrude B. Elion of Burroughs Wellcome Laboratories for determinations of plasma 4,6-diVOL.
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Homeostasis-Steele,
Rieselbach
875
hydroxypyrozolopyrimidine and oxypurine levels and to Dr. S. T. Bloomfield of Burroughs Wellcome and Co. for generous supplies of Zyloprim. REFERENCES 1. NUGENT, C. A. and TYLER, F. N. Renal excretion of uric acid in patients with gout and in nongouty subjects. J. Clin. Znuest., 38: 1890, 1959. 2. RIESELBACH, R. E., BENTZEL, C. J., COTLOVE, E., FREI, E. and FREIREICH, E. J. Uric acid excretion and renal function in the acute hyperuricemia of leukemia. Am. J. Med., 37: 872, 1964. 3. GUTMAN,A. B., Yi?, T. F. and BERGER, L. Tubular secretion of urate in man. J. Clin. Znuest., 38: 1778, 1959. 4. GUTMAN, A. B. and Yti, T. F. Three-component system for regulation of renal excretion of uric acid in man. Tr. A. Am. Physicians, 74: 353, 1961. 5. BERLINER, R. W., HILTON, J. G., Yii, T. F. and KENNEDY, T. J., JR. Renal mechanism for urate excretion in man. J. Clin. Invest., 29: 396, 1950. 6. WEINER, J. M. and MUDGE, G. H. Renal tubular mechanisms for excretion of organic acids and bases. Am. J. Med., 36: 743, 1964. 7. SWTH, H. W. Principles of Renal Physiology, p. 196. New York, 1956. Oxford University Press. 8. ROE, J. E., EPSTEIN, J. H. and GOLDSTEIN, N. P. A photometric method for the determination of inulin in plasma and urine. J. Biol. Chem., 178: 839,1949. 9. LIDDLE, L., SEEGMILLER, J. E. and LASTER, L. The
IO.
11.
12.
13.
14.
15.
16.
enzymatic spectrophotometric method for determination of uric acid. J. Lab. & Clin. Mfd., 54: 903,1959. ELION, G. B., KOVENSKY, A., HITCHINGS, G. N., METZ, E. and RUNDLES, R. W. Metabolic studies of allopurinol, an inhibitor of xanthine oxidase. Biochem. Pharmacol., 15: 863, 1966. DAVIS, B. B., FIELD, J. B., RODNAN, G. P. and KEDES, L. H. Localization and pyrazinamide inhibition of tubular secretion of uric acid-C?” with modified stop-flow technique. J. Clin. Invest., 43: 1281, 1964. Yii, T. F., BERGER, L., STONE, D. J., WOLF, J. and GUTMAN,A. B. Effect of pyrazinamide and pyrazinoic acid on urate clearance and other discrete renal functions. Proc. Sac. Exper. Biol. &’ Med., 96: 264, 1957. GUTMAN,A. B. and Yii, T. F. Renal function in gout; with commentary on renal regulation of urate excretion, and role of kidney in pathogenesis of gout. Am. J. Med., 23: 600, 1957. SEEGMILLER, J., GRAYZEL, A., HOWELL, Ii. and PLATO, C. Renal excretion of uric acid in gout. J. Clin. Znuest., 41: 1094, 1962. Yii, T. F., BERCER, L. and GUTMAN, A. B. Renal function in gout. II. Effect of uric acid loading on renal excretion of uric acid. Am. J. Med., 33: 829, 1962. WYNGAARDEN,J. B. Discussion. In: Proceedings of Conference on Gout and Purine Metabolism, p. 683. New York, 1965. Grune & Stratton, Inc.
Mechanism
for Urate
in Normal THOMAS H. STEELE,
M.D.~
Homeostasis
Man*
and RICHARD
E.
RIESELBACH,
M.D.
Madison, Wisconsin In thirty-two studies on ten normal subjects, renal urate secretion and reabsorption have been estimated by noting the decrement in UVurate: Clnulin produced by a maximally effective dose of pyrazinamide (TS,,), a drug which markedly although probably not completely inhibits tubular urate secretion. In each subject studies were performed in the control state, after decreasing plasma urate with allopurinol, and following elevation of plasma urate levels by RNA loading. Urate reabsorption remained at an average of 98 per cent of filtered load at all plasma levels, thereby indicating progressive augmentation of reabsorptive transport velocity with increasing filtered loads. Also, tubular secretion per nephron was a direct function of plasma urate concentration. Although plasma urate levels ranged as high as 12.8 mg. per 100 ml., at no time was there evidence for attainment of a secretory or reabsorptive tubular transport maximum. A provisional normal standard was set forth, relating TS,, to plasma urate concentration through a regression treatment with appropriate confidence limits. These data suggest that normal man displays an augmented rate of bidirectional urate transport with increasing substrate availability. Due to the nature of this bi-
directional transport system, an increase in secretory rate serves to increase net urate excretion and thereby provides the homeostatic mechanism which tends to minimize the hyperuricemic response to an increase in uric acid synthesis. The pyrazinamide suppression technic appears to provide a means of assessing the integrity of this homeostatic mechanism within the residual nephrons of the chronically diseased kidney or in patients who have gout. cult in that the renal excretion of urate is governed via a bidirectional transport system. There is now convincing evidence that filtered urate is virtually completely reabsorbed; that urate which appears in the final urine apparently gains access predominately through active secretion [3,4]. Studies performed to date suggest that at least a twofold increase in plasma urate concentration would be necessary to exceed the maximum rate of tubular reabsorption (T,) for urate [5]. Therefore, an increase in filtered load of urate, secondary to an increased plasma urate concentration, would not serve as a regulatory mechanism until marked hyperuricemia supervened unless the reabsorptive mechanism exhibited a marked degree of splay.
normal man excessive ingestion of uric acid precursors usually results in a significant increase in the rate of urinary urate excretion in association with only a modest increase in the plasma urate concentration [I]. Similarly, in some myeloproliferative disorders minimal elevations in plasma urate concentration may be accompanied by marked increases in the urinary excretion of urate [2]. Thus the human kidney may significantly increase its rate of urate excretion in the presence of even a modest degree of hyperuricemia, thereby serving to minimize the hyperuricemic response to ex*;enously or endogenously derived urate loads. Characterization of the mechanism underlying this augmentation of excretory rate is diffi-
I
N
* From the Department of Internal Medicine, The University of This study was supported by U. S. Public Health Service Research and The Wisconsin Alumni Research Foundation. Requests for Rieselbach, M.D., Department of Medicine, University Hospitals, 53706. Manuscript received December 16, 1966. t Postdoctoral Research Fellow, U. S. Public Health Service. Center, U.S. Public Health Service Hospital, Baltimore, Maryland.
868
Wisconsin Medical School, Madison, Wisconsin. Grants AM 09943-01, AM 05630, FR 5435-05 reprints should be addressed to Richard E. 1300 University Avenue, Madison, Wisconsin Present
AMERICAN
address:
Baltimore
JOURNAL
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MEDlClNE
Renal
Mechanism
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It appears more reasonable to examine the secretory component of the bidirectional transport system for urate. There is evidence to suggest that urate may share the same secretory mechanism as other organic acids such as paraaminohippurate [6]. Therefore, it is reasonable to postulate that, just as for para-aminohippurate, the velocity of mate transport from renal peritubular fluid to tubular lumen should increase as substrate availability increases, until a maximum is reached where intrinsic tubular factors might become rate limiting. In the present studies an attempt has been made to examine the rate of urate secretion per nephron in ten normal control subjects by observing the change in the urinary excretion of urate per unit of glomerular filtration following suppression of urate secretion by the administration of pyrazinamide. By conducting a minimum of three separate studies on each subject at different plasma urate levels (attained by pharmacologic manipulation), data were obtained which confirm the presence of a sensitive regulatory mechanism for urate within the kidney. This system contributes to urate homeostasis through an alteration of secretion rate in response to changes in the plasma urate level. An attempt has been made to quantify this relationship so that the appropriate secretory response to a given plasma urate level may be defined within predicted limits in normal man. MATERIALS
AND METHODS
A total of thirty-two studies were performed in ten normal volunteer subjects. This group was composed of eight male physicians and one male and one female patient from the general medical service. None of the subjects had evidence of renal disease on the basis of history, physical examination or laboratory findings. All subjects were Caucasian except for V.W., who was of Oriental extraction. Each subject underwent a minimum of three studies. Each study consisted of two phases; the first phase involved at least three clearance periods of fifteen to thirty minutes in duration. Following this 3 gm. of pyrazinamide was given orally. After an interval of approximately one hour, the first of three additional clearance periods of similar duration was initiated. The majority of subjects exhibited maximum suppression of urinary urate excretion in either the second or third period after the administration of pyrazinamide. During the three clearance periods following the dose of pyrazinamide, however, the rate of urinary urate excretion did not vary by more than 10 per cent from the average of the baseline periods. Therefore, it seemed likely that maximal suppression VOL.
43,
DECEMBER
1967
Homeostasis-Steele,
Rieselbach
was being observed within the time interval employed, and that carrying out the clearance procedure for a longer time would be of little value. Standard clearance technics were employed [7] ; urine flow of 7 to 15 ml. per minute was induced by aggressive oral hydration so that all clearance studies could be carried out without catheterization. Each subject was studied at a minimum of three different plasma urate levels. The first study was performed at that subject’s endogenous plasma urate level. Then allopurinol* (4-hydroxpyrazolopyrimidine) was administered for seven days at a dose of 600 to 800 mg. per day in order to reduce the plasma urate concentration by decreasing the rate of urate synthesis through xanthine oxidase inhibition. The second study was carried out after allopurinol had been discontinued for twenty-four hours in order to allow time for excretion of the drug, its primary metabolite, alloxanthine (4,6-dihydroxypyrazolopyrimidine), and the oxypurine byproducts of enzyme inhibition. The third study was performed a minimum of one week following the second study. Prior to this study 4 gm. of yeast ribonucleic acid (RNA) was administered daily for four days. In two subjects a fourth study was carried out following further RNA or intravenous uric acid loading. Glomerular filtration rate was determined by the clearance of inulin; inulin analyses were carried out according to the method of Roe et al. [a]. Uric acid determinations were performed by the enzymatic apectrophotometric method of Liddle et al. 191. RESULTS
Estimation of Tubular Secretion of Urate ( TS,,) by the Pyrazinamide Suppression Technic. In every study the administration of pyrazinamide resulted in a sharp decrease in urinary urate excretion. Plasma urate levels were essentially unchanged from the first to the second phase of each study. Because of the desirability of expressing excretion relative to a given population of nephrons, urate excretion values are factored by the inulin clearance, thus giving an estimate proportional to urate excretion per nephron. t In the representative study illustrated in Table I, inulin clearance and plasma urate level remained quite constant. The mean excretion of urate during the control periods was 4.23 pg. per minute per glomerular filtration rate (or pg. per ml.). Fifty-eight minutes following the oral administration of 3 gm. of pyrazinamide, excretion * Zyloprim@, Burroughs Wellcome 56-l 58.
t Hereafter, for the sake of brevity, the term excretion per nephron wiil be employed interchangeably with excretion per milliliter of glomerular filtrate. However, it is to be understood that the two are not synonymous, but that the former is merely proportional to the latter, and is of much smaller magnitude in absolute terms.
870
Renal
Mechanism
for Urate
Homeostasis-Steele,
TABLE PROTOCOL
I
OF A REPRESENTATIVE SUBJECT
Rieselbach
STUDY
W.S.
C
Cinulin (ml./min.)
0 43 61 81 101 Mean values
Begin clearance
159
TS,,
(mg.Ikf?ml.)
C (ml.~rZn.)
Cinulin
FE,,,, (estimate
-
administration;
1.0 1.3 1.7 1.34
4.2 4.4 4.2 4.23
7.0 7.2 7.1 7.11 empty bladder
0.51 0.66 0.80 0.657
0.90 1.1 1.4 1.15
uvurste decrease in Cinulin induced by pyrazinamide
= maximum = (4.23)
periods after pyrazinamide 5.7 5.8 5.6 5.71
116 111 122 116
Cinulin (rg/mi.)
(%)
Inulin prime and sustaining infusion Begin clearance periods; empty bladder 8.7 5.9 124 9.0 6.1 126 9.1 5.8 128 8.96 5.94 126 Pyrazinamide, 3 gm. given orally
179 195 211 Mean values
NOTE:
UVurate __
2%x~oo
Time (min.)
(0.51)
= maximally of fraction
= 3.72 pg./min./Ci,,ii, (estimate of urate secretion C depressed Cir:,tL ~ after pyrazinamide = 0.90 per cent.
per nephron).
of filtered urate escaping reabsorption).
per nephron (UV,,,,, :Cinulin) had decreased to 0.51 pg. per ml. Although this is the minimum level of excretion observed under secretory inhibition, the values during consecutive time intervals are similar. In order to assess urate secretion as quantitatively as possible, the minimum value of excretion (at maximal drug effect) was compared to the control mean excretion value. In this particular study UVurate :Cinulin decreased 88 per cent. If this decrease in urate excretion occurred as a result of secretory inhibition, then tubular secretion must account for at least 88 per cent of urinary urate excretion in this subject at a plasma urate concentration of 5.9 mg. per 100 ml. Tubular secretion of urate per nephron (TS,,) is then calculated as the mean initial excretion per nephron, 4.2 pg. per ml., minus excretion per nephron at maximal pyrazinamide effect, 0.51 pg. per ml. The result 3.7 pg. per ml., is regarded as an estimate of the secretory rate per unit of glomerular filtration rate, and hence is proportional to the secretory rate per nephron. In addition to control studies, all subjects were restudied on different occasions at a minimum of two other plasma urate concentrations. Plasma urate was decreased through xanthine oxidase inhibition by allopurinol, or increased through the administration of yeast RNA.
The results of all studies are given in Table II. In every subject a decrease in plasma urate level resulted in both decreased total urate excretion and urate secretion per nephron. Likewise, elevation of the plasma urate concentration was accompanied by elevated excretion and secretion rates. Response of Tubular Reabsorptive Rate to Change in Filtered Load of Gate. A summary of all the data (Table III) indicates that the percentage decrease in urate excretion following the administration of pyrazinamide was essentially the same, irrespective of the plasma urate level. Thus, at all three plasma concentrations secretion appeared to constitute a relatively stable percentage of excretion, averaging approximately 80 per cent. Since it is reasonable to consider that the decrement in urate excretion produced by pyrazinamide occurs solely through inhibition of secretion, any remaining urate excretion during maximal action of pyrazinamide reflects the amount of filtered urate which has escaped reabsorption during passage through the nephron, if suppression of tubular secretion of urate by pyrazinamide is assumed to be complete. Hence, fractional urate excretion (FE,,.&,) = nonreabsorbed filtered urate : total filtered Simiurate = Curate:Cinuiin after pyrazinamide. larly, an estimate of the fraction of filtered urate AMERICAN
JOURNAL
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Renal
Mechanism
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Homeostasis-Steele,
reabsorbed would be 11 - (Curute:Cinur,nafter pyrazinamide)]. From Table III, it may be seen that the mean FE,,,,, approximates 2 per cent of filtered load at all plasma mate levels. Furthermore, in individual subjects (Table II) FEurat, is a relatively stable quantity irrespective of plasma urate concentration. Thus, urate reabsorption was proportionally augmented with increasing filtered loads (Fig. 1) ; a reabsorptive T, was not reached with the plasma urate concentrations employed in these studies. Response of Urate Secretory Rate to Changes in Plasma Urate Concentration. As shown in Table increased or decreased appropriately II, TS,, with induced changes in plasma urate concentraTABLE COMPLETE
DATA
Rieselhach
871
tion in all patients studied. At a mean plasma urate of 5.35 mg. per 100 ml., mean TS,, was 4.01 pg. per ml. (Table III). Lowering of plasma urate in all subjects to a mean of 3.26 mg. per 100 ml. after allopurinol administration resulted in a decrease of mean TS,, to 1.93 pg. per ml. Elevation of plasma urate to a mean of 8.40 mg. per 100 ml. after RNA loading resulted in an augmentation of mean TS,, to 8.40 pg. per ml. Determinations of 4,6-dihydroxypyrazolopyrimidine levels were carried out in three subjects by Dr. Gertrude Elion of BurroughsWellcome Co., employing an isotope dilution method [lo]. These subjects had received 800 mg. of allopurinol per day for one week, and at II
ON TEN NORMAL
SUBJECTS
Secreted Urate Age (yr.) and Sex R.H.,
Cinulin
(ml./min./ 1.73 M2.)
32, M
(113)
T.S. 29, M
(119)
G.S., 41, F
(114)
V.W.,
(101)
32,M
W.S., 29, M
(110)
R.R.,
M
(106)
A.M., 28, M
(107)
A.K.,
30, M
(108)
F.G., 29, M
(96.9)
R.C.,
(117)
32,
26, M
C “r*ta
Plasma Urate
Patient,
Cinulin x 100
Study* Control HPP RNA (1) RNA (2) Control HPP RNA (1) RNA (2) Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA Control HPP RNA
(%) 5.8 3.8 9.0 12.8 5.8 4.3 7.9 12.2 3.8 2.5 5.3 4.3 2.8 6.3 5.9 3.4 9.6 4.8 3.1 9.1 4.8 3.7 7.4 6.1 2.8 7.0 6.4 3.3 7.1 5.6 2.9 7.1
518 215 920 1,458 598 299 695 1,853 560 402 808 670 339 1,279 470 222 942 355 202 1,402 350 272 679 583 261 1,068 539 269 1,021 769 218 906
7.0 4.8 10.3 10.6 8.8 5.4 7.5 14.0 14.5 14.1 11.9 16.7 11.3 19.4 7.1 5.8 9.3 6.6 6.5 14.4 8.0 7.4 7.1 8.6 8.3 15.3 9.0 8.1 15.2 11.8 6.7 10.6
* HPP = studies performed after one week of 4-hydroxypyrazolopyrimidine formed after four days of ribonucleic acid administration. VOL.
43,
DECEMBER
1967
TS,, (pg./ml.) 2.7 1.3 7.8 9.8 4.3 1.9 5.0 15.5 2.9 1.3 3.8 6.1 3.0 11.7 3.7 1.8 8.2 2.5 1.7 10.3 3.6 2.4 4.7 4.1 2.1 9.3 4.9 2.3 8.7 5.2 1.5 6.2 administration;
(%)
Excreted Urate x 100 (70)
2.6 1.5 1.6 3.0 1.4 0.9 1.1 1.3 6.6 9.1 4.7 2.7 0.8 0.9 0.9 0.2 0.9 1.3 1.2 3.1 0.6 0.8 0.8 1.8 0.6 2.1 1.2 1.3 2.8 2.6 1.8 1.9
65.7 70.7 84.5 72.3 84.2 83.0 84.9 91.2 52.2 36.5 60.2 84.7 92.8 95.1 87.9 95.3 91.1 80.1 83.3 78.6 92.5 89.2 89.3 78.7 92.2 86.6 86.2 85.2 80.2 79.0 75.9 82.8
FEu,ate X 100
RNA
= studies per-
Renal Mechanism
872
for Urate
Rieselbach
Homeostasis-Steele,
TABLE III SUMMARY OF DATAIN TABLE II IIPP Data
Cinulin
(ml./min./l.73 Plasma urate (mg./lOO ml.) UV”&? (pg./min.) C (yy- x 100
Control
RNA
Mean
S.E.
Mean
S.E.
Mean
S.E.
111 .o
(2.9)
108.0
(3.9)
110.0
(3.3)
Ms.) 3.26 270.0
(0.17) (20.0)
5.35
(0.27)
8.40
(0.66)
541 .o
(41 .O)
1,087.O
(101 .O)
12.1
(1.05)
7.83
(0.91)
9.80
(1.08)
TS”,~
1.93
(0.17)
4.01
(0.37)
8.40
(0.96)
(P&/ml.) (FL.t.) X 100
1.83
(0.82)
2.15
(0.54)
2.01
(0.34)
,Il”IllJ
(%)
(%)
(Secreted
Urate)
(Excreted
Urate) >
(%)
x
80.4
100
(5.5)
79.1
(3.8)
83.1
NOTE: HPP = studies performed after one week of 4-hydroxypyrazolopyrimidine administration. performed after four days of ribonucleic acid administration. See text for definition of parameters.
the time of study, twenty-four hours after stopping administration of the drug, values ranged from 0.78 to 0.88 mg. per 100 ml. The plasma oxypurine level in one subject at this time, also determined by Dr. Elion, was 0.1 mg. per 100 ml. Although definitive studies in man have not been carried out as yet it is unlikely that these levels of drug metabolite and enzyme inhibition by-product, respectively, influenced urate secre120 100 60 60
FILTERED URATE
: G,,,,,
(JW/ml.)
FIG. 1. The proximity of the points to the d:agonal line of complete reabsorption at all plasma urate levels indicates virtually complete reabsorption of filtered urate (mean 98 per cent throughout the range of filtered load studied). TR,,,, = P,,.,. (I - Curate: Cinurinafter pyrazinamide administration), a minimal estimate of urate reabsorption per nephron.
(2.8)
RNA
= studies
tion or excretion to a significant degree. Thus, in each subject the only variable was the plasma urate concentration. Therefore, it is reasonable to ascribe the change in secretory rate to variations in this parameter. Predictability of Urate Secretion in Relation to Plasma Urate Concentration. One goal of these studies has been to derive a standard nomogram which would establish the normal range of urate secretion (TS,,) at a given plasma urate concentration. The data presented in Table II reveal that the secretory response to a given plasma urate level is not identical from one subject to another. Figure 2 (tok), in which TS,, is plotted as a function of plasma urate, graphically illustrates the wide range of secretory response encountered in five subjects. Two subjects (T.S. and R.H.) who had received additional RNA and sodium urate intravenously, respectively, exhibited acceleration and deceleration of TS,,, respectively, at plasma urate levels over 12 mg. per 100 ml. One subject (V.W.) never became hyperuricemic after RNA loading, presumably because of a brisk rate of urate secretion (TS,, 11.7 pg. per ml. at a plasma urate of 6.3 mg. per 100 ml.). Another (A.K.) exhibited brisk acceleration of TS,, as soon as the hyperuricemic state was attained. With one exception (V.W.), values in all subjects tended to cluster TS,, together during studies at normal and decreased plasma urate levels. AMERICAN
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Renal
Mechanism
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Figure 2 (bottom) depicts urate to inulin clearance ratios in the same five subjects, expressed as functions of plasma urate concentration. The relationship between TS,, and C&,: C inulin is inconstant because of the artifact introduced into the latter by factoring excretion per nephron by plasma urate [Curate: Cinuiin = is uvurate : Cinuiin X 1 : PuratP]. Although TS,, a nearly linear function of plasma urate in R.C., C urate: C in” iin actually decreases upon attainment of hyperuricemia. Likewise, Curate: Cinuiin remains constant in R.H. as the uppermost value of plasma urate (12.8 mg. per 100 ml.) is approached. Thus, since parallelism between is not always mainC “rate: Cinuiin and TS,, tained, the former parameter appears to be poorly descriptive of urate secretion. In attempting to relate TS,, to plasma urate, separate linear regression treatments of studies at normal and elevated plasma urate levels
873
Rieselbach
Homeostasis-Steele,
(excluding G.S. and V.W., two subjects who did not achieve a plasma urate over 6.5 mg. per 100 ml. after RNA loading) revealed values of standard error of the estimate of 1.073 and 2.443, respectively. The variance of TS,, is significantly greater at hyperuricemic levels (F = 5.145; P < 0.01) than at lower plasma urate concentrations. Therefore, a linear regression treatment of TS,, and plasma urate, with common variance, is not justified. A log-log regression treatment of all data is depicted in Figure 3. The resulting nonlinear regression obtained is concave upward and has a gradual increase in variance as TS,, and P,, increase. In Figure 3 the 95 per cent confidence limits of the regression itself, and the 95 per cent prediction confidence limits for individual data, are depicted graphically. Although the prediction confidence limits could be narrowed somewhat by eliminating subject V.W. from the
PLASMA URATE (mg/l~mt.)
16
II
2
I,
4
I,
6
Ill
I,
8
10
12
PLASMA URATE (mq/lOOml.)
I
I 2
I
I 4
I
I
,
6
P~JYDM URATE
I 8
,
I IO
,
1
,
12
(mg./lOOmL)
FIG. 2. Top, TS,,
is plotted as a function of plasma mate concentration in five subjects. Note the wide range of secretory response observed at each level of plasma mate. Bottom, C”,., : Cinul in plotted as a function of plasma mate concentration for the same five subjects. Note the inconstant relationship of Curate :Cio”lin to the actual rate of mate secretion (TS,,). VOL.
43,
DECEMBER
1967
Fig. 3. Log-log regression treatment of TS,, on plasma mate in thirty-two studies on ten normal subjects, replotted on a linear scale. The regression equation, TS,, = 0.413 (plasma mate)‘.%9 is indicated along with the correlation coefficient. The 9.5 per cent confidence limits of the regression (f0.0559 log units) are indicated by the darkly shaded area. The 95 per cent confidence limits for prediction of normalcy (f0.331 log units) are indicated by the lightly shaded area. The one point lying outside the 95 per cent prediction confidence limits is that of V.W. after RNA loading. Note that this subject did not achieve hyperuricemia because of a brisk secretory response. Variance of the secretory rate per nephron (TS,,) is significantly increased in hyperuricemia (see text).
874
Renal Mechanism
for Urate
regression, there is no definite justification
for removing these data from the study. Thus the TS,, of a subject would not be considered distinctly abnormal unless it happened to lie outside the 95 per cent prediction confidence interval of Figure 3. COMMENTS
A technic employing the administration of a large, single dose of pyrazinamide has been utilized as a means of quantitatively estimating the component of renal urate excretion which is derived from an active tubular secretary process. In addition, if complete filtration of urate at the glomerulus may be assumed, an estimate of urate reabsorption within the nephron was obtained. Because of the probability that complete inhibition of the secretory mechanism was not attained with this technic, the estimates obtained are those of minimal secretion and reabsorption at a given plasma urate level. Admittedly these data are based upon the assumption that the decrease in excretion of urate following the administration of pyrazinamide results from suppression of secretion, as suggested by stop-flow studies in the dog [II]. It is difficult to conceive that pyrazinamide could act by virtue of enhancing the rate of urate reabsorption, although there is no evidence in man to rule out this possibility unequivocally. The similar degree of apparent secretory suppression between different subjects and between individual studies in the same subject suggests that this technic is of adequate reproducibility. The dose of 3 gm. of pyrazinamide is more than employed in most previous studies in man [ 721; this dosage level was established in order to minimize variations in response which might occur due to different rates of drug absorption within individual patients. Although this technic is not completely quantitative, we believe that it represents a significant advance in assessing the renal mechanism for urate excretion. Most previous studies have attempted to evaluate the renal excretory capacity for urate by employing the parameters of UVurate:Ginuiin and Gurate:Ginuiin ]7,73,74]. Both parameters would appear to be nonspecific because they represent the resultant effect of two independent processes, as previously pointed out by Yti et al. [75]. The urate to inulin clearance ratio is particularly lacking in physiologic significance. Although this parameter gives the ratio of excreted to filtered urate, the pyrazinamide suppression data reveal that a maximum
Homeostasis-dteele,
Rieselbach
of only 2 per cent of filtered urate is excreted on the average. Thus, since the bulk of urinary urate is derived from tubular secretion, there appears to be no justification for factoring the amount excreted by the filtered load. This parameter might correlate reasonably well with TS,, in some patients, but the present data suggest that the correlation is quite unpredictable and is not sufficiently quantitative to serve as a means of comparing the renal response to hyperuricemia in any group of patients (Fig. 2.). The application of the technic described herein considerably increases the specificity of information which may be derived from studies of urate excretion, as may be further emphasized by the following example. It has already been noted that urate reabsorption is nearly complete within the normal nephron with mean values of FEurate of approximately 2 per cent at all plasma levels studied. Two subjects (G.S. and V.W.) had control urate to inulin clearance ratios of 14.5 and 16.7 per cent, respectively; on this basis each would formerly have been classified as having a “reabsorptive defect for urate.” However, the FEurate value in one (G.S.) was 6.6 per cent, whereas in the other (V.W.) it was only 2.7 per cent. The increased excretory rate of the latter subject was due to a markedly increased rate of secretion rather than a deficient rate of reabsorption. Thus, this technic allows estimation of secretion and reabsorption as independent events-each with definable normal limits. The formulation of TS,, involves the utilization of glomerular filtration rate as a basic index of nephron population. As long as the average glomerular filtration rate per nephron remains relatively constant, the total glomerular filtration rate will reflect the number of functioning nephrons. Therefore, the expression of urate corrects for losses in secretion “per nephron” the functioning nephron population incurred through disease, and thus provides a means of evaluating the performance of nephrons within the context of their functional mass at a given point in time. In addition, voiding errors become less critical because of the use of only urine and plasma concentrations in the calculations. It has been suggested previously by Wyngaarden [76] that renal urate secretion, expressed as a function of plasma urate level, may describe a sigmoidal pattern which initially accelerates with increasing plasma urate levels, and then reaches a plateau as a hypothetical tubular secretory maximum is approached. On the basis of our data we can only conclude that AMERICAN
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OF
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Renal
Mechanism
for Urate
such a secretory maximum must occur only at very high plasma urate levels. However, over the range of plasma urate concentrations employed in the present studies, the data might be considered to be consistent with the kinetics of an enzyme system displaying a sigmoidal velocitv-substrate relationship. ‘These data suggest that normal man is capable of augmenting both reabsorptive and secretory transport in the face of increased substrate availability. Theoretically, by employing sufficient urate loading and utilizing pyrazinamide a reabsorptive and secretory T, suppression, should be demonstrable. The marked augmentation of urate reabsorption with increased filtered loads serves to protect the nephron from an overwhelming load of urate in the presence of hyperuricemia. However, the secretory component of this bidirectional transport system appears simultaneously to attain an increased rate of activity. The net effect is a comparatively moderate increase in urate excretion. Nevertheless, the degree of hyperuricemia resulting from the accession of a given quantity of urate to the extracellular space is modified, and a reasonable degree of homeostasis is maintained. As an extension of the foregoing thesis the question arises as to the integrity of these transport systems in disease. The experimental technic described should provide a means of determining whether these transport systems remain intact within the residual nephrons of the chronically diseased kidney, thereby enabling these units to respond appropriately to a given degree of hyperuricemia and effectively contribute to homeostasis. Also, this technic should provide a precise means of characterizing the renal mechanism for urate excretion in gout. If the etiology of gout in some patients is clearly associated with a deficient rate of urate secretion, it is reasonable to expect that TS,, in these patients at hyperuricemic and pharmacologically induced normouricemic levels should distinctly differ from that of normal subjects. The great variability of TS,, at elevated plasma urate levels in normal subjects emphasizes the need for well defined normal limits of excretory rate at a given plasma urate level if the excretory mechanism in patients with gout is to be compared to that of normal subjects. Acknowledgment: We are indebted to Dr. Gertrude B. Elion of Burroughs Wellcome Laboratories for determinations of plasma 4,6-diVOL.
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hydroxypyrozolopyrimidine and oxypurine levels and to Dr. S. T. Bloomfield of Burroughs Wellcome and Co. for generous supplies of Zyloprim. REFERENCES 1. NUGENT, C. A. and TYLER, F. N. Renal excretion of uric acid in patients with gout and in nongouty subjects. J. Clin. Znuest., 38: 1890, 1959. 2. RIESELBACH, R. E., BENTZEL, C. J., COTLOVE, E., FREI, E. and FREIREICH, E. J. Uric acid excretion and renal function in the acute hyperuricemia of leukemia. Am. J. Med., 37: 872, 1964. 3. GUTMAN,A. B., Yi?, T. F. and BERGER, L. Tubular secretion of urate in man. J. Clin. Znuest., 38: 1778, 1959. 4. GUTMAN, A. B. and Yti, T. F. Three-component system for regulation of renal excretion of uric acid in man. Tr. A. Am. Physicians, 74: 353, 1961. 5. BERLINER, R. W., HILTON, J. G., Yii, T. F. and KENNEDY, T. J., JR. Renal mechanism for urate excretion in man. J. Clin. Invest., 29: 396, 1950. 6. WEINER, J. M. and MUDGE, G. H. Renal tubular mechanisms for excretion of organic acids and bases. Am. J. Med., 36: 743, 1964. 7. SWTH, H. W. Principles of Renal Physiology, p. 196. New York, 1956. Oxford University Press. 8. ROE, J. E., EPSTEIN, J. H. and GOLDSTEIN, N. P. A photometric method for the determination of inulin in plasma and urine. J. Biol. Chem., 178: 839,1949. 9. LIDDLE, L., SEEGMILLER, J. E. and LASTER, L. The
IO.
11.
12.
13.
14.
15.
16.
enzymatic spectrophotometric method for determination of uric acid. J. Lab. & Clin. Mfd., 54: 903,1959. ELION, G. B., KOVENSKY, A., HITCHINGS, G. N., METZ, E. and RUNDLES, R. W. Metabolic studies of allopurinol, an inhibitor of xanthine oxidase. Biochem. Pharmacol., 15: 863, 1966. DAVIS, B. B., FIELD, J. B., RODNAN, G. P. and KEDES, L. H. Localization and pyrazinamide inhibition of tubular secretion of uric acid-C?” with modified stop-flow technique. J. Clin. Invest., 43: 1281, 1964. Yii, T. F., BERGER, L., STONE, D. J., WOLF, J. and GUTMAN,A. B. Effect of pyrazinamide and pyrazinoic acid on urate clearance and other discrete renal functions. Proc. Sac. Exper. Biol. &’ Med., 96: 264, 1957. GUTMAN,A. B. and Yii, T. F. Renal function in gout; with commentary on renal regulation of urate excretion, and role of kidney in pathogenesis of gout. Am. J. Med., 23: 600, 1957. SEEGMILLER, J., GRAYZEL, A., HOWELL, Ii. and PLATO, C. Renal excretion of uric acid in gout. J. Clin. Znuest., 41: 1094, 1962. Yii, T. F., BERCER, L. and GUTMAN, A. B. Renal function in gout. II. Effect of uric acid loading on renal excretion of uric acid. Am. J. Med., 33: 829, 1962. WYNGAARDEN,J. B. Discussion. In: Proceedings of Conference on Gout and Purine Metabolism, p. 683. New York, 1965. Grune & Stratton, Inc.