Clinical Science (1994) 86, 227-231 (Printed in Great Britain)
Gender differences in urinary kallikrein excretion in man:
variation throughout the menstrual cycle
J. D. M. ALBANO, S. K. CAMPBELL, A. FARRER and 1. G. B. MILLAR
Department of Renal and Endocrine Medicine, University of Southampton, St Mary's Hospital,
Portsmouth, U.K.
(Received 20 August 1992/22 July 1993; accepted 16 August 1993)
1. Urinary kallikrein excretion was measured in
healthy male subjects and in healthy pre- and postmenopausal females.
2. Urinary kallikrein excretion was shown to be
constant throughout a 24h period. Individual male
subjects showed little fluctuation in urinary kallikrein
excretion; within-subject variance accounted for
1.65% of the total.
3. Female subjects with ovulatory menstrual periods
excreted significantly more kallikrein than postmenopausal females and males.
4. Pre-menopausal females showed a much greater
within-subject variation in urinary kallikrein excretion
and this could be related to the stage of the menstrual cycle, with significantly greater urinary kallikrein excretion in the luteal phase than in the
follicular phase.
5. Plasma renin activity and plasma aldosterone
concentration also showed a menstrual variation, with
concentrations in the luteal phase being significantly
higher than those in the follicular phase.
6. The rise in urinary kallikrein excretion in the
luteal phase could be abolished by oral administration
of the aldosterone antagonist spironolactone.
7. Urinary kallikrein excretion in post-menopausal
females was similar to the range found in males, and
showed no cyclic changes over a 4 week period.
8. Gender and menstrual status should be taken into
account in studies of the physiological role of tissue
kallikreins.
INTRODUCTION
Renal kallikrein is thought to play an important
role in the regulation of blood pressure and water
and electrolyte excretion, by local generation of
vasodilatory kinins and stimulation of prostaglandin
biosynthesis [ 1, 21. However, measurements of prostaglandins and tissue kallikrein and kinins in peripheral blood have failed to account for their apparent actions, and a paracrine role for the kallikreinkinin system has been proposed [3]. The excretion
of kallikrein in urine is believed to reflect intrarenal
production, and it is found in both active and
inactive forms [4, 51. Urinary kallikrein excretion
may be altered by manipulation of mineralocorticoid status, either by modification of dietary
sodium or administration of a synthetic sodiumretaining steroid [6, 71. In addition urinary kallikrein excretion has been reported in cases of clinical
hyperaldosteronism [8, 91. Reduced urinary kallikrein excretion has been found in hypertensive
animal models [lo, 111 and in some forms of
human hypertensive disease [9, 123. It has been
proposed that reduced renal kallikrein may play an
important part in the development of hypertension
because of the failure of this vasodilatory system
adequately to oppose the vasoconstrictor reninangiotensin-aldosterone (RAA) system. In man, not
all studies have found urinary kallikrein excretion to
be different in normotensive and hypertensive subjects [13, 141. Although factors such as race, sodium
status, renal function and age have been investigated
[15, 161, less attention has been paid to gender. In
1953, exogenous kallikrein was reported to lead to
irregularities within the menstrual cycle and to
increase libido in males [17], indicating possible
interactions between the kallikrein-kinin system and
reproductive hormones. Gender-related differences
in the tissue kallikrein content of the submaxillary
and pituitary glands have been reported in rats, and
these differences have been related to oestrogen
status [18, 191. In a human study, females have
been shown to excrete more kallikrein than males,
but menstrual status or phase of the cycle was not
reported [20].
In this study, we have assessed urinary kallikrein
excretion in healthy male and female subjects, in
relation to circadian changes of posture and the
RAA system, and, in females, to the stage of the
menstrual cycle. The effect of the aldosterone antagonist, spironolactone, on menstrual variation in
urinary kallikrein excretion has been studied in premenopausal females.
METHODS
Healthy male and pre-menopausal females (age
range 22-39 years) and post-menopausal females
Key words gender differences, menstrual cycle, spironolactone. urinary kdlikrein.
Abbreviations PA. plasma aldortemne concentration; PRA, plasma renin activity; MA.reni~angiotensiMldmterone; SP, serum prqerterone concentration.
Correspondence: Dr 1. D. M. Albano. Department of Renal and Endocrine Medicine, University of Southampton, St Mary's Hospital. Portsmouth PO3 6AD, U.K.
228
J. D. M. Albano et al.
(age range 52-58 years), were sequentially studied
over an 8 week period. The pre-menopausal female
subjects had regular menstrual cycles (26-30 days),
and the post-menopausal women had not had a
period for 2 years. All subjects were normotensive
with no history of renal disease or evidence of
dysfunction, as indicated by normal serum creatinine concentration and absence of urinary tract
infections as judged by N-Labstix analysis (Bayer
Diagnostics). The four subjects intensively studied
had creatinine clearances within normal limits.
None of the subjects was receiving any medication
and no dietary restrictions were imposed. The study
was approved by the ethical subcommittee of the
Portsmouth and South East Hampshire District
Health Authority.
Twenty-four hour urine collections were made,
with no preservative and were subdivided into fractions as follows: 08.0&12.00 hours, 12.W16.00
hours, 16.W20.00 hours and 20.0&08.00 hours. All
samples were assayed for kallikrein activity within 5
days. Varying numbers of samples were collected
from each subject. In the case of males, the interval
was random; in the females, collections were made
on day 7 + 2 days of the menstrual cycle representing the follicular phase and on day 2 2 k 2 days
representing the luteal phase. Blood samples were
collected on these days for determination of plasma
renin activity (PRA), plasma aldosterone concentration (PA) and serum progesterone concentration
(SP), between 09.00 and 10.00 hours with the subjects in the seated position, after approximately 2 h
of ambulation.
Since results obtained from the first part of this
study showed urinary kallikrein excretion to be
remarkably constant throughout the 24 h period (see
Fig. Ib), four female subjects made more frequent
urine collections throughout a complete cycle, but
collections were made from 20.00 to 08.00 hours. In
addition, six female subjects each made 2 x 12h
overnight collections on successive days during the
follicular and luteal phases of three consecutive
cycles. Spironolactone (Aldactone; Searle Pharmaceuticals), 100mg daily, was taken from day 14 of
the second cycle until day 22 of the third cycle. S P
was measured during follicular and luteal phases of
all cycles. Urine was kept in a cool place during
collection, and aliquots were stored at 4"C, and
assayed within 5 days of collection. Kallikrein-like
activity was measured using the chromogenic substrate S2266 (Kabi Diagnostica) by a modification
of the method of Amundsen et al. [21]. Tissue, but
not plasma kallikrein, splits the substrate H-D-ValLeu-Arg-pNA, and the p-nitroaniline (pNA) released
is directly proportional to the amount of kallikrein
present. All measurements of urinary kallikrein
excretion reported in this study refer to kallikrein in
the active form. The assay was standardized using
purified porcine kallikrein (BNLI/80 Bayer UK,
Ltd). The intra-assay coefficient of variation was
2.3% ( n = 12) and the inter-assay coefficient of
1
T
08.W12.00 12.W16.00
16.00-20.00
2O.W-OB.00
Time of day (hours)
I
0
I
I
I
I
20
40
60
80
i
100
Daytime kallikrein excretion (m-unitsih)
Fig. I. Urinary kallikrein excretion. (a) Urinary kallikrein excretion
throughout the day (08.00-20.00 hours, 3 x 4h collection periods) and
overnight ( 2 0 . W . 0 0 hours). Values are means fSEM (n = 40). (b)
Relationship between day time (08.W20.00 hours) and night-time
(20.GO-OE.00 hours) urinary excretion of kallikrein. The correlation
coefficient for linear regression analysis is 0.98 (P<O.001, n = U ) .
variation was 3.1%. Urinary kallikrein excretion rate
was expressed as m-units/h. PRA was measured by
a modification of the method of Haber et al. [22],
PA by the method of Al-Dujaili and Edwards [23]
and SP by routine radioimmunoassay. Results contained in the text are expressed as meansfSD,
mean fSEM and correlations by linear regression
analysis. Statistical analysis was by Student's t-test
using Bonferroni protection.
RESULTS
Sub-division of the 24h collections made on all
subjects into 4 h periods during the day, and 12h
overnight collection showed no significant variation
in urinary kallikrein excretion throughout the 24 h
period (Fig. la). A close correlation (r=0.98,
Gender differences in kallikrein excretion
229
..
7
t
t
-' +
i
I
v
0 :
i
Group I
Group 2
Group 3
Group 4
Fig. 2. Urinary kallikrein excretion (24 h collections). Group I, males,
n=32; group 2, post-menopausal females, n= 14; group 3. ovulating
females (follicular phase), n=27; group 4, the same females (luteal phase),
n = 22. Horizontal bars represent the mean for each group.
P < 0.001; n = 46 by linear regression analysis) was
obtained between nocturnal and diurnal urinary
kallikrein excretion in both sexes (Fig. lb). The
mean value of urinary kallikrein excretion for males
was 27.6 f20.9.4 m-units/h (range 4-85 m-units/h,
n = 32). Within-subject variance accounted for only
1.65% of the total variance, indicating little fluctuation in the daily excretion rate of kallikrein in
males.
When compared with male subjects, a higher
daily excretion of kallikrein was seen in ovulating
females (mean 43.4 f25.4 m-units/h, range 9-90 munits/h, n = 49, P < 0.003). In addition, females
showed a much greater within-subject variance in
kallikrein excretion, and this was subsequently
shown to reflect regular fluctuation over the menstrual cycle. On day 7 the daily excretion was
21.7f5 (n=27) m-units/h and on day 22 it was
70.2 k9.6 (n= 22) m-units/h (P<0.OOOl). A similar
pattern was seen in PRA with values of 1.5+ 1.2
(n=9) and 4.3k1.8 (n=8)ng of angiotensin I
h-'ml-' on days 7 and 22, respectively (P<O.OOl).
Values for PA were 227k74.9 ( n = 11) and
413f89.3 ( n = 10)pg/ml, respectively (P<O.OOOl).
Urinary kallikrein excretion in post-menopausal
females was 20.6 9.3 m-units/h (n = 14) and did not
differ significantly from concentrations found in the
follicular phase of ovulating females or from values
found in the male subjects (Fig. 2). Furthermore,
when these subjects were re-studied over a 4 week
period there was no evidence of cyclic fluctuations.
In the four subjects in whom more frequent
collections were made, urinary kallikrein excretion
showed a marked rise around and after the time of
ovulation from 23f4.4 on day -22 to 56k9.2 on
day -3 (P<O.OOl). A sharp fall in urinary kallikrein excretion to 32.6 L 10.2 m-units/h occurred
before the onset of menstruation (Fig. 3). Ovulation
lo
o
1l
l
l
l
l
l
l
l
l
-28-26-24-22-20-18-16-14-12-10
Day of cycle
,
,
l
- 8 - 6 -4
l
l
-2
l
0
Fig. 3. Urinary excretion of kallikrein (20.W8.W hours) in four
subjects throughout the menstrual cycle. The onset of menstruation is
plotted as day 0. and ovulation was confirmed by measurement of SP.
was confirmed in all untreated cycles by SP concentrations > 22 nmol/l in the luteal phase.
Urinary kallikrein excretion in the follicular and
luteal phase of the control cycles of the six subjects
in the spironolactone study were 21.6f 5.2 and
42.3 f9.6 m-units/h, respectively. For the first cycle
of spironolactone urinary kallikrein excretion in the
follicular phase was 19 k 3.1 m-units/h, and was not
significantly different from that in the control cycle.
Values in the luteal phase in this cycle were
9.5 f2.3 m-units/h and showed a significant reduction (P<O.OOl) when compared with the same
phase in the control cycles. During the second cycle
on spironolactone, urinary kallikrein excretion in
the follicular phase was 14.0 & 5.4 m-units/h (five
subjects) and in the luteal phase was 15.1 k6.6munits/h (four subjects) (Fig. 4). One subject discontinued spironolactone after the first cycle, while in
another subject cycle length was reduced by 14
days. During the second cycle on spironolactone
four of the six subjects reported a shortening (3, 3, 4
and 14 days) of previously regular cycles. For each
subject, luteal phase SP concentrations were
increased over follicular phase values, but due to
shortening of cycles these may not have been peak
concentrations. All cycles lengths returned to
normal when the drug was stopped.
DISCUSSION
The RAA system has been extensively studied in
relationship to blood pressure and electrolyte
balance in health and disease, and its importance is
established and well documented. By contrast, the
opposing vasodilatory kallikrein-kinin system is less
well understood, although previous studies have
established a connection between mineralocorticoid
activity and urinary excretion of kallikrein [7]. In
J.
D. M. Albano et
~
O
’
FLI
LI
F12
L2
FL3
L3
Fig. 4. Urinary kallikrein excretion (20.0048.W hours) during
Cllicular (FL) and l u t u l (1)phases in six subjects throughout three
consecutive menstrual cycles. Each point represents the mean of t w o
samples collected on consecutive days The hatched area represents the
period of rpironolactone administration, and the horizontal bars the mean
for each group All SP values were >22mmol/l during L2 In one subject
( 0 )cycle length was reduced t o 14 days during the second cycle on
rpironolactone. but returned to 28 days in the first cycle after spirone
lactone with an SP of 39mmol/l at day 22 and a urinary kallikrein excretion
of am-unitsih
this study we have shown urinary kallikrein
excretion to be relatively constant throughout a 24 h
period, in contrast to the activity of the R A A
system, which shows clear circadian variation as a
result of postural changes [24]. Furthermore, male
subjects and post-menopausal females showed little
variation in urinary kallikrein excretion when they
were re-studied over several weeks. This was in
contrast to ovulating female subjects, who showed
clear-cut menstrual variation in urinary kallikrein
excretion; that in turn could be related to both
ovarian hormones and mineralocorticoid activity.
Whether the increases in active urinary kallikrein
excretion during the luteal phase represent an
increase in kallikrein synthesis, or a change in
specific activity due to conversion of inactive to
active kallikrein, is not clear. The availability of a
radioimmunoassay to measure immunoreactive
kallikrein, or measurement of urinary kallikrein
activity after tryptic activation, would shed further
light on these observations. In addition, we have
confirmed previous findings of a clear-cut variation
in the activity of the RAA system through the
menstrual cycle [25].
Evidence from studies in uitro supports the view
that spironolactone, which is similar in structure to
aldosterone, acts at the cellular receptor level as a
competitive inhibitor of this mineralocorticoid
action on sodium transport. In addition, spironolactone is also believed to affect steroid biosynthesis
and, although the precise point of the steroidogenic
pathway involved has not been clearly identified,
impairment of 1 1 - and 18-hydroxylase activities has
al
been implicated [26]. Studies in male and female
subjects have shown spironolactone administration
to result in a partial inhibition of aldosterone
secretion, but
SP concentrations
remained
unchanged [27, 281. Although progesterone exhibits
weak natriuretic and anti-mineralocorticoid activity,
the abolition of the rise in urinary kallikrein
excretion in the luteal phase by spironolactone in
our subjects is consistent with reports, in males,
showing a strong association between mineralocorticoid activity and urinary kallikrein excretion [7].
In males, the side effects of spironolactone treatment, such as gynecomastia and impotence, have
been ascribed to anti-androgenic properties due to
competitive interaction with nuclear and cytoplasmic receptors for dihydrotestosterone, and progestogenic properties due to structural similarities [29].
Although, in females, menstrual irregularities have
been reported on a dose of 400mg of
spironolactone/day [27, 301, such effects were unexpected at the much lower dose of l00mg/day used
in our study. It is therefore possible that the
progestogenic and anti-androgenic actions of
spironolactone disturb the menstrual cycle in a yet
unexplained way.
Studies in rats have shown that renal tissue
kallikrein and renal kallikrein mRNA levels are
higher in ovulating females than in males [31, 321,
and our studies in man showing overall greater
excretion of kallikrein in females are in accord with
these observations. A role for ovarian hormones,
therefore, in renal kallikrein production cannot be
excluded and may explain the reduced urinary
kallikrein excretion in post-menopausal women.
The lack of a circadian variation in urinary
kallikrein excretion would exclude a direct and
immediate effect of changes in PA; this contrast
between the absence of circadian variation in
urinary kallikrein excretion and the presence of
menstrual variation may reflect a relatively slow
response of urinary kallikrein excretion to changes
in mineralocorticoid activity. Studies of urinary
kallikrein excretion during administration of exogenous mineralocorticoid would support this suggestion [7]. A rise in the activity of the kallikrein-kinin
system after activation of the R A A system could
have the effect of ‘damping’ the increase in blood
pressure and preventing ‘overshoot’.
In this study, we have confirmed a gender difference in the excretion of kallikrein in man, and have
shown that this difference reflects the differing patterns of gonadal hormone secretion and mineralocorticoid activity. Equivocal reports of reduced
urinary kallikrein excretion in human hypertension
may therefore be partly explained by gender differences, as well as differences in methodologies for
kallikrein determination. We conclude that gender
and the stage of the menstrual cycle should be
considered in further studies of the physiological
role of tissue kallikreins, particularly in relation to
hypertension and renal disease.
Gender differences in kallikrein excretion
ACKNOWLEDGEMENT
We thank Mr D. Kirk, Michael Darmady
Laboratories, Queen Alexandra Hospital, Cosham,
Hants, U.K., for measurements of SP.
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