A V Brooks

J E Baer

Karl H Beyer

R M Noll

CiteSeerX

8823
319
EFFECT OF HYDROCHLOROTHIAZIDE ON RENAL ELECTROLYTE
GRADIENT IN GLUCOSE DIURESIS AND EXPERIMENTAL
DIABETES INSIPIDUS
J. E. BAER, A. V. BROOKS, R. M. NOLL AND KARL H. BEYER
Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania
Received for publication May 9, 1962
Hydrochlorothiazide and other thiazides are
very effective in increasing the elimination of
salt in animals and in man. The saluresis may
be accompanied by extensive water diuresis,
especially in subjects with fluid retention, but it
has been demonstrated in several studies that
thiazides do not specifically increase the “free
water” clearance (Heinemann ci at., 1959;
Blackmore, 1959). On the other hand, Crawford
and Kennedy (1959) and Kennedy and Craw-
ford (1959) have shown that in experimental
and clinical diabetes insipidus, thiazides will
actually diminish urine flow while rendering the
urine more hypertonic.
The present experiments were designed to
relate the disparate action of thiazides on sodium
and on water excretion in normal and diabetes
insipidus animals to the current concepts of
renal transport of water. The countercurrent
multiplier theory which has been proposed to
explain the renal tubular reabsorption of water
is based in part on the observation that a renal
gradient exists such that the sodium concentra-
tion is extremely high in the inner medulla. The
effects of hydrochlorothiazide, experimental dia-
betes insipidus and glucose infusion on the renal
sodium gradient in dogs and rats are presented
in this paper.
METHODS. Female mongrel dogs, weighing about
9 kg, stabilized on a laboratory regimen for at
least 1 week, were given 500 ml of water by gavage,
then anesthetized with pentobarbital (30 mg/kg).
In Group I dogs, one kidney was removed through
a flank incision, then hydrochlorothiazide (50 or
100 mg/kg) was given as a single dose i.v., and
23/i hours later the second kidney was removed.
In Group II dogs, one kidney was removed, then
5% glucose was infused at 10 ml/min for 23/i
hours prior to removal of the second kidney. In
Group III, 5% glucose (10 ml/min) was infused
starting 30 minutes prior to removal of the con-
trol kidney. After removal of the first kidney,
hydrochiorothiazide (100mg/kg) was incorporated
in the glucose infusion which was continued for
23/i hours prior to removal of the second kidney.
Kidneys were refrigerated at once, and precisely
defined sections of cortex, outer medulla and
inner medulla were removed promptly. Weighed
portions of tissue were digested in 1:5 nitric acid
at 70#{176}Cfor several hours and diluted appro-
priately for measurement by internal standard
flame photometry. Excellent agreement was ob-
tained when duplicate specimens from any zone
were digested separately and analyzed (table 1).
In some cases dry weight data were obtained, but
as the interpretation of results remained the same,
wet weight data only are shown here.
Two pairs of 250-gram rats with experimental
diabetes insipidus (kindly supplied by Dr. John
Crawford) were stabilized on a restricted food
intake and ad lib.water intake supplemented by
intubations with 5 ml of water three times daily.
One pair then was given 45 mg/kg day of hydro-
chlorothiazide for 3 days as a suspension in water.
On the fourth day, all rats were sacrificed 2 hours
following a dose of 5 ml of water (or 15 mg/kg of
hydrochlorothiazide in water). Within 5 minutes
of decapitation, sections of papilla and cortex,
and portions of medulla freed from cortical
tissue but including cortical-medullary junc-
tion, were separated. Tissues were transferred
to tared beakers, weighed and dried at 70#{176}C
for 72 hours. The dry tissues were weighed, di-
gested for 24 hours at 80#{176}Cwith 5 ml of 1.5 N
nitric acid and diluted appropriately for the de-
termination of electrolyte content. Four normal
rats were given an identical dietary regimen with
water ad lib. supplemented by thrice daily intuba-
tion of 5 ml of water as above and kidney speci-
mens were taken for comparison as unoperated
controls.
The effect of chlorothiazide (100 mg/kg twice
daily) was likewise examined in rats with experi-
mental diabetes insipidus; these rats were surgi-
cally prepared in the authors’ laboratory by the
technique of Crawford and Kennedy (1959).
320 BAER ET AL. Vol. 137
TABLE 1
Sodium and potassium content of dog kidneys
removed before (control) and .5 hours after
intravenous injection of hydrochiorothiazide
(Group I)
Dog 45 Dog 410 Dog 415t
Na1 K1 Na1 K Na K1
m Eq per kg wet weight of tissue
Control
Plaama (mEqJI) - - 146 3.2 140 3.6
Cortex 68 62 67
69
56
55
66
73
64
62
Outer medulla 186 45 149
145
45
47
134
142
39
39
Inner medulla 195 39 185
156
36
42
151
141
45
47
After hydrochloro-
thiazide
Plaema (mEqJ1) - - 143 2.4 133 2.9
Cortex 61 61 65
64
57
61
67
72
55
56
Outer medulla 170 45 167
157
42
56
155
156
38
38
Inner medulla 191 51 196
194
46
55
141
136
50
46
Excretion, mEq/2.5 hr - - 24.5 5.4 21.4 16.4
0 Dog 4 5 received 50 mg/kg of hydrochlorothiazide and no
water loading. No plaama or urine aamplea were collected.
t In Dog 4 15, hydrochiorothiazide plaama concentration wae
78 mg/I 2 hours after drug adminiatration; 40% of injected drug
was excreted in 24 hours.
RESULTS. Group I. Hydrochlorothiazide alone
had no effect on the wet weight sodium or
potassium content of cortical or medullary regions
of Group I dogs (table 1). Data are presented for
3 dogs; similar results were obtained in a fourth
animal. The saluretic effect of the drug was
made evident by the excretion of more than 20
mEq of sodium by Dogs 10 and 15. In agree-
ment with previous studies, the sodium content
in the medullary regions of the control kidneys
was much higher than that in the cortex, and
was equal to or greater than that of plasma. In
every dog, the electrolyte content in each seg-
ment of the control kidney agreed very closely
with that in the kidney removed 2 hours
following the administration of hydrochloro-
thiazide.
Group II. The renal sodium gradient was
again clearly seen in the control kidneys of
Group II dogs, the cortex being hypotonic and
the medulla very hypertonic (table 2).
After infusion of 5% glucose for 2 hours,
the gradient was nearly obliterated. The wet
weight sodium content of the outer medulla was
reduced from 170 mEq/liter to 70 mEq/liter.
That of the inner medulla was reduced from
225 mEq/liter to 70 mEq/liter. The cortical
sodium and potassium concentrations, initially
hypotonic, were further reduced by glucose
infusion. The medullary potassium concentra-
tion was not changed.
Group III. The control kidneys from these
dogs pretreated for  hour with 5% glucose,
contained a much lower concentration of sodium
than those of Group I, especially in the medulla
(table 3). The medullary sodium was not as low
as in Group II, wherein glucose had been infused
for a longer time. In spite of the low renal sodium
content, the venoclysis of hydrochlorothiazide
again caused the excretion of more than 20 mEq
of sodium.
The sodium content of the second kidney of
glucose-infused dogs, removed after hydro-
chiorothiazide administration, was somewhat
lower than that in the control kidneys (table
3). Similar results were obtained in 3 additional
dogs not included in the table. In each of the
comparable tissues, the wet weight sodium
content of the second was 10 to 40 mEq/liter
lower than its corresponding control. The values
in all cases closely resembled those obtained
TABLE 2
Obliteration of renal electrolyte gradient 2.5 hours
following the infusion of 5% glucose at 10
mi/mm to dogs (Group II)
Dog *11 Dog 412 Dog *16’
Na K1 Na1 K1 Na1
m Eq per kg wet weight of tissue
Control
Plasma (mEq/l) 150 4. 1 150 4.2 140 3.5
Cortex 65 64 68 69 81
81
51
51
Outer medulla 168 39 181 44 180
172
43
42
Inner medulla 223 44 227 44 196
193
39
39
After glucose infusion
Plasma (mEq/1) 116 3.1 108 3.8 116 3.0
Cortex 41 45 47 40 50
53
45
46
Outer medulla 71 - 69 32 81)
86
38
34
Inner medulla 65 41 72 40 78
75
38
43
Excretion, mEq/2.5 hr 6.2 1.5 5.0 2.4 2.0 1.8
* Two different specimens from each region were analyzed.
TABLE 4
Sodium and water content in kidneys of rats
mEq er kg wet weight of tissue
Control-0.5 hr after beginning 5% glucose venoclysis
Plasma (mEq/l) 121 2.9
Cortex 85 47 82 52 57 46
Outer medulla 117 46 126 55 110 37
Inner medulla 79 38 106 48 148 42
After hydrochlorothiazide-5% glucose venoclysis maintained
for 2.5 hours
Plasma (mEq/l) 105 3.1
Cortex 56 45 66 41 44 42
Outer medulla 87 38 67 44 71 32
Inner medulla 61 36 66 41 64 34
Excretion, mEqJ2.5 hr 27.5 5.1 29.2 3.7 13.7 2.5
Unoperated’
Diabetes insipidus,t no
drug
%
Water
Papilla
82
78
81
80
78
87
86
86
86
83
86
87
* In Dog * 19, hydrochlorothiazide plasma concentration
was 51 mg/I 2 hours after drug administration was started; 22%
of injected drug was excreted in 2 hours.
89
94
76
65
129
180
156
131
383
722
368
236
1962 HYDROCHLOROTHIAZIDE AND RENAL GRADIENT 321
TABLE 3
Lack of effect of hydrochiorothiazide (100 mg/kg,
iv.) on “washed out” renal electrolyte gradient
of glucose infused dogs (Group III)
Dog *2 Dog *4 Dog *19’
Na’ K1 Na1 K’ Na1 K4
after the infusion of glucose alone for 2  hours
(Group II). This supports the view that the
Group III controls were not quite equilibrated
and that the apparent drug effect may be merely
a result of the prolonged infusion of glucose in
this group. Potassium content was not affected
by the hydrochlorothiazide.
Rats. A pronounced sodium gradient existed
in the kidneys of rats in experimental diabetes
insipidus (tables 4 and 5). In spite of the marked
polyuria and polydipsia (urine volumes ranged
from 105 to 172 mi/day) the renal sodium
gradient was not obliterated. In fact, the mean
wet weight sodium content in these kidneys was
not significantly different from that of un-
operated control animals which received the
same diet.
Following the administration of hydrochioro-
thiazide, polyuria and polydipsia diminished
within 24 hours, and electrolyte excretion di-
minished somewhat more slowly over a 3-day
period. In these animals, upon autopsy, the
renal sodium gradient was also present (table 4).
The wet weight sodium contents were not
statistically distinguishable from those in the
unoperated rats or in the diabetic animals with
polyuria.
The water content of renal medullary tissue
appeared somewhat higher in both groups of
diabetes insipidus animals than in the unoperated
Diabetes insipidus,
hydrochlorothiazidef
15mg/kg t.i.d.X 3 days
Na1, mEajkg Wet
Weight of Tissue
Me- Pa-ortex dulla pilla
60 125 297
64 142 362
62 160 297
65 133 418
87 142 477
78 131 502
69 138 396
69 137 552
* Individual rats, average of kidney pair.
f Individual kidneys.
TABLE 5
Sodium and water content in kidneys of rats with
experimental diabetes insipidu8
Na1, mEqjk Wet
Weight of Tissue
Cortex Jia 
%
Water
Papilla
Diabetes insipidus, no
drug
Diabetes insipidus,
chlorothiazide, 100
mg/kg b.i.d. X 3 days
71
67
67
70
77
67
61
62
116
147
198
153
135
147
134
140
414
304
585
377
525
681
361
508
80
81
87
79
82
86
81
86
rats (ca. 84% vs. ca. 80%); this difference would
be reflected if a “dry” weight sodium content
were calculated. Because of the very small tissue
weights, especially of the papillae, the differences
are not believed to be significant.
Chlorothiazide was likewise given to one pair
of rats with experimental diabetes insipidus;
the daily water intake fell from 145 ml (average
of 3 days pretreatment) to 25 and 30 ml on the
third day of chlorothiazide administration. The
322 BAER ET AL. Vol. 137
sodium content of the kidney regions from these
animals (table 5) demonstrates a marked gm-
dient. The gradient did not differ appreciably
from that of a matched pair of control animals,
although the latter exhibited marked polyuria
and polydipsia throughout the experimental
period prior to sacrifice.
DIscussIoN. Wirz et al. (1951) demonstrated
an osmotic pressure gradient that increased
progressively from the renal cortex to reach a
maximal value at the tip of the medullary papilla.
This gradient has been interpreted as being due
to the functioning of the loop of Henle in the
manner of a countercurrent multiplier system
wherein the active transport of sodium from the
lumen of the ascending limb contributed critically
to the phenomenon and to the resultant gradient,
as reviewed by Smith (1959). Malvin and Wilde
(1959) showed that when dogs were submitted
to 20% mannitol venoclysis at rates sufficient to
increase urine flow to 10 to 15 mi/mn this con-
centration gradient was obliterated. Previously
Ullrich et al. (1955) and Ullrich and Jarausch
(1956) and later Levitin et al. (1961) showed that
the osmotic and sodium gradients were reduced
in dogs following water or sulfate diuresis. Malvin
and Wilde (1959) considered that rate of flow
through the loops was too great to permit
efficient operation of the countercurrent mech-
anism, and referred to this situation as a “wash-
out” of the renal countercurrent gradient.
It seems certain that under relatively mild
conditions of 5% glucose venoclysis at 10 ml/min
some such factor as the increased flow through
the loop of Henle is sufficient to abolish the
electrolyte gradient from cortex to medulla, as
illustrated in table 2. On the other hand, the
administration of hydrochlorothiazide in a dosage
sufficient to inhibit maximally both proximal
and distal reabsorption of sodium does not
influence the sodium gradient from cortex to
medulla, notably (table 1). This being the case,
it was anticipated that the hydrochlorothiazide-
induced increase in sodium presented to the
countercurrent system of the loop might serve
to re-establish the glucose-depleted sodium
gradient from cortex to inner medulla. This
was not the case, within the limits of these
experiments wherein the glucose venoclysis was
maintained (table 3). Viewed differently, the
data indicate that the saluretic effect of hydro-
chiorothiazide was not dependent on the main-
tenance of the sodium gradient (table 3).
Sufficient data were obtained to demonstrate
that hydrochlorothiazide was exerting a pro-
nounced saluretic effect during the experiments,
but relationships between urine flow, osmolar
clearance and water reabsorption were not
examined in this study. Earley et al. (1961) have
shown that chlorothiazide may cause a relative
decrease in water reabsorption in unanesthetized
dogs under conditions of extreme diuresis, and
have proposed that an important aspect of the
action of thiazides is located in the distal nephron.
It is interesting that the increasing osmolar
gradient from cortex to inner medulla is only
slightly lower than normal in rabbit (Crawford,
1957), the hamster (Gottschalk, 1961) or the
rat (table 4) having an induced state of diabetes
insipidus. Somehow, hydrochlorothiazide is capa-
ble of decreasing the polyuria and the polydipsia
of experimental diabetes insipidus (Crawford and
Kennedy, 1959) without influencing notably (by
this methodology) the gross sodium concentra-
tion gradient across the kidney (table 4).
These two types of experiments may be inter-
preted to indicate that neither saluretic nor the
antidiuretic effect of hydrochiorothiazide in.flu-
ences discernibly the sodium gradient from
cortex to inner medulla; nor is the saluretic effect
of the thiazide dependent on the maintenance of
that gradient as by a countercurrent multiplier
system.
SUMMARY
Hydrochlorothiazide (100 mg/kg, i.v.) did not
affect the renal electrolyte gradient in unin.fused
dogs.
The renal electrolyte gradient was obliterated
by infusion of 5% glucose alone (10 ml/min).
Renal medullary sodium fell from 225 to 68
mEq/liter. The gradient was not re-established
upon concurrent administration of hydrochloro-
thiazide.
An essentially normal renal sodium gradient
was present in rats with experimental diabetes
insipidus. Polyuria and polydipsia were sup-
pressed by chlorothiazide or hydrochlorothiazide
with no change in the renal sodium content.
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CRAWFORD, J. D. AND KENNEDY, G. C.: Nature,
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EFFECT OF HYDROCHLOROTHIAZIDE ON RENAL ELECTROLYTE GRADIENT IN GLUCOSE DIURESIS AND EXPERIMENTAL DIABETES INSIPIDUS

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EFFECT OF HYDROCHLOROTHIAZIDE ON RENAL ELECTROLYTE GRADIENT IN GLUCOSE DIURESIS AND EXPERIMENTAL DIABETES INSIPIDUS

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