Clinical Science (1993) 84, 11-19 (Printed in Great Britain)
Stimulatory action of parathyroid hormone on renin
secretion lfn vitro: a study using isolated rat kidney,
isolated rabbit glomeruli and superfused dispersed rat
juxtaglomerular cells
Christian SAUSSINE, C. JUDES, T. MASSFELDER, M.-J. MUSSO, U. SIMEONI, T. HANNEDOUCHE
and J.-J. HELWIG
laboratoire de Physiologie Cellulaire Renale, Universite Louis Pasteur, Strasbourg, France
(Received 15 April/7 September 1992; accepted 10 September 1992)
1. We have previously reported that pharmacological
concentrations (125 nmol/l) of parathyroid hormone
may stimulate renin release in the stable recirculating
and non-filtering isolated rat kidney.
2. In the present study we have attempted to extend
these initial observations by examining the
concentration-related response of renin release to
parathyroid hormone, using the same model of isolated kidney, and determining whether the effect of
parathyroid hormone on renin release can be demonstrated by more direct approaches. Thus, the effects
of parathyroid hormone on renin secretion were
investigated in two other renal preparations: isolated
rabbit glomeruli and isolated rat juxtaglomerular
cells.
3. In the isolated kidney, rat parathyroid hormone
significantly stimulated renin accumulation in the
perfusate in a concentration-related manner with a
threshold of 1nmol/l.
4. In both glomeruli and juxtaglomerular cells bovine
[Nle" 18,Tyr34]parathyroid
hormone-(1-34)amide
effectively and repeatedly stimulated renin release.
These results imply that there is a direct stimulatory
effect of parathyroid hormone on renin release.
5. We also examined the effect of [NIe'*'',
T ~ r ~ ~ l p a r a t h y r o i dhormone-( 1-34)amide
during
extracellular calcium buffering in the glomeruli.
[Nle8~'8,Tyr34]parathyr~id
hormone-(l-34)amide
was uneffective in calcium-free medium. Increasing
the extracellular ionized calcium concentration to
2.5 mmol/l increased the extent of stimulation in
accordance with the reported ability of parathyroid
hormone to block calcium channels and relax vascular smooth muscle cells.
6. These results provide further support for the role
of parathyroid hormone as a direct mediator of renin
secretion; moreover, the renin-stimulating action of
parathyroid hormone may be mediated through the
inhibition of calcium influx.
INTRODUCTI0 N
Based upon a variety of pharmacological and
physiological evidence, it has become increasingly
clear that parathyroid hormone (PTH), in addition
to its hypercalcaemic properties, relaxes contractile
activity in isolated vascular tissues [l-S] and
increases renal blood flow [9-111. Recently, we
demonstrated that bovine or rat (r) PTH can induce
a concentration-related relaxation of the vascular
bed of the preconstricted isolated rat kidney [12].
The adenylate cyclase system could be involved in
this renal vascular action since PTH stimulates
adenylate cyclase activity in a rabbit renal microvessel preparation enriched with glomerular arterioles [13-151. PTH also binds with high affinity to
those vessels in a specific and saturable manner
[161. These physiologically relevant influences of
PTH on the renal vascular bed raise the question of
whether PTH may also influence other functions of
the renal vascular smooth muscle cells, such as
renin secretion from juxtaglomerular (JG) cells,
which are modified smooth muscle cells [17-191. In
support of an action of PTH on renin secretion, it
has been shown that PTH can block calcium entry
in smooth muscle cells of the rat tail artery [20, 211.
Also, the proposal that factors which stimulate renin
release do so by lowering the cytosolic calcium
concentration or increasing the cellular cyclic AMP
concentration has received considerable support
during the past few years [22, 231.
Exogenous PTH was previously found to produce
a consistent rise in plasma renin activity in humans
and dogs [24-281. However, these studies did not
clarify whether this represented a direct action of
PTH on the JG cells or not. Effects mediated by
either an activation of the baroreceptors resulting
from renal vasodilatation [12] and/or a sodium
chloride load at the macula densa are other factors
postulated to alter renin release [22, 26, 291.
Key words: angiotensin I assay, calcium, isoprenaline, parathyroid hormone, renin secretion, verapamil.
Abbreviations: ANG I, angiotensin I; JG, juxtaglomerular; KRBG, Krebs-Ringer bicarbonate glucose buffer (for composition, see the text); NlePTH, [Nle8.l8, Tyr"1parathyroid
hormone-( I-34)amide; PMSF, phenylmethanesulphonyl fluoride; PTH, parathyroid hormone; r, rat.
Correspondence: D r 1.1. Helwig, Laboratoire de Physiologie Cellulaire Renale, Pavillon Poincare. H8pital Central, BP 426, 67091 Strasbourg Cedex. France.
I2
C. Saussine et al
In a recent study, we found that pharmacological
concentrations (125 nmol/l) of rat PTH-( 1-34) stimulated renin release in the isolated recirculating
rat kidney, perfused in non-filtering conditions, at
constant flow and stabilized pressure, by a mechanism independent of alterations in systemic and
renal haemodynamics and macula densa sodium
chloride concentrations [30]. This suggested a direct
action of PTH on the JG cells.
The aim of the present work was to further
extend these findings. We first intended to establish
the concentration-related response of renin release
to PTH, using the same model of the isolated
kidney. We then attempted to reproduce the stimulating action of PTH on renin release, using isolated
glomeruli and superfused collagenase-dispersed cortical cells enriched in JG cells. These approaches
should help to better segregate extracellular
influences on renin release from the intracellular
events. In the three different assay systems in uitro,
we found that PTH produced a consistent and
direct stimulation of renin release.
MATERIALS AND METHODS
Chemicals
PTH-( 1-34) and bovine [Nle8s'8,Tyr34]PTH(1-34) amide (NlePTH) were purchased from Bachem
Feinchemikalien AG (Bubendorf, Switzerland). The
peptides were dissolved in 1 mmol/l HCl and, 1 mg/
ml BSA at a concentration of 0.25mmol/l and
stored at - 70°C in 25 pl portions. BSA (fraction V)
was obtained from Euromedex (Schiltigheim,
France) and was dialysed overnight against KrebsHenseleit buffer. (-)-Isoprenaline
and ( +)verapamil were purchased from Sigma (St Louis,
MO, U.S.A.), and L-ascorbic acid from Merck
(Darmstadt, Germany). [3-['251]iodotyrosine-4]angiotensin I was from Amersham International
(Amersham, Bucks, U.K.). Angiotensin I (ANG I)
was from Peninsula Laboratories (Belmont, CA,
U.S.A.). Rabbit anti-ANG I was from Calbiochem
(San Diego, CA, U.S.A.). Collagenase (type 11) for
renal cortex cell dispersion was purchased from
Worthington (Freehold, NJ, U.S.A.) Collagenase
(type I) for glomeruli preparation was from Sigma.
Percoll and Sephadex G- 15 were from Pharmacia
(Uppsala, Sweden). Sodium pentobarbital was from
Clin-Midy (St Jean de la Ruelle, France).
Perfusion of isolated rat kidney
Male Wistar rats (170-220 g body weight), with
free access to standard pellet food and tap water,
were used. Kidney perfusion was performed according to the basic description of Schurek et al. [31]
with some modifications [l2, 15, 301. In brief, on
the day of experiment the animals were anaesthetized by intraperitoneal injection of sodium
pentobarbital (45 mg/kg body weight). After opening
of the abdominal cavity the right kidney was
exposed and placed on a thermostatically controlled
metal cupel. After an intravenous injection of
heparin (5 units/g body weight; Choay, Paris,
France) and ligation of the suprarenal aorta, perfusion through the superior mesenteric artery was
started without ischaemia with an initial flow rate of
about 5 ml/min. Thereafter, the kidney was isolated
from the animal and was perfused in a recycling
perfusion circuit by draining the renal venous
effluent, via a cannula placed in the vena cava, back
into a thermostatically controlled (37°C) recirculating reservoir. The perfusate volume was 50ml and
was continuously gassed with 95% 0,/5% CO,. The
kidney preparations were rendered non-filtering by
the use of perfusate with a high oncotic pressure
(lOOmg/ml BSA), a low perfusion pressure
(70mmHg) and by ligation of the ureter by the
technique of Maack [32]. Perfusion flows, expressed
as mlmin-' g - ' of contralateral kidney, were
adjusted to achieve a perfusion pressure of
70mmHg during the first 10-15min using a peristaltic pump (Minipuls 3; Gilson, Villiers-le-Bel,
France), and remained constant for the rest of the
experiment, which lasted up to 80min. The mean
perfusion flow applied to reach 70 mmHg was
19.8ml min- ' g- (SEM 0.8; n = 25). The kidney
preparations in which the perfusion flows were
lower than 12 ml min-' g-' were systematically
rejected. The perfusion pressure was continuously
monitored by a potentiometric recorder (type
Physiocardiopan; Philips, Paris, France) through a
pressure transducer (Statham P23Db; Statham Labs
Inc., Hato Rey, Puerto Rico) placed in the infrarenal
aorta. The basic perfusion medium, which was taken
from the recirculating reservoir, consisted of a
Krebs-Henseleit bicarbonate solution containing
5 mmol/l glucose, 6 mmol/l urea, 1 mmol/l sodium
oxaloacetate, 5 mmol/l sodium lactate, 2 mmol/l
sodium pyruvate and all physiological amino acids
in concentrations between 0.5 and 5mmol/l. The
perfusate was supplemented with 100 mg/ml BSA.
Ionized calcium concentrations in the medium and
pH were determined in a Ca2+/pH analyser (CibaCorning Diagnostic CO, Medfield, MA, U.S.A.) and
were adjusted to 1 mmol/l and 7.4, respectively.
The time course of renin activity accumulating in
the recirculating perfusate of kidneys receiving
rPTH-( 1-34) at 38 min was compared with separate
time-control kidneys receiving no PTH. The time
intervals between the sampling points were identical.
Perfusate samples of 5Opl were collected at 15min
intervals, beginning at 35min, up to 80min. The
samples were cooled at -20°C and were diluted 25
times before being assayed for renin activity.
'
Incubation of isolated rabbit glomeruli
Rabbit (2-2.2 kg) renal cortex glomeruli were
prepared using a collagenase dispersion of the cor-
Direct action of parathyroid hormone on renin release in vitro
tex, followed by sieving steps through calibrated
stainless-steel sieves to remove all tubule and microvessel fragments. The collagenase dissociation of the
rabbit renal cortex was performed exactly as described previously [14]. The dissociated cortex was
suspended in ice-cold Krebs-Ringer bicarbonate
glucose buffer (KRBG). This buffer contained (in
mmol/l): NaCl, 120; KC1, 4.6; MgCl,, 0.5;
Na2HP04, 0.7; NaH,PO,,
1.5; glucose, 10;
NaHCO,, 15. The ionized calcium concentration
and pH of the KRBG were adjusted to 1.5mmol/l
and 7.4, respectively, as described above, and the
buffer was gassed continuously with 95% 0,/5%
CO,. The dissociated cortex in KRBG was filtered
stepwise through a stainless-steel sieve of 125 pm
pore size. This filtration removed most of the long
tubular fragments. The 125pm filtrate was then
filtered 15 times through a 70pm sieve. These
filtrations gradually removed the microvessels and
the residual tubule fragments, and the final retained
material was purified glomeruli. Multiple 5 pl aliquots of a well-mixed suspension were removed for
measurement of glomeruli number by counting in a
standard haemocytometer at 40 x power under light
microscopy and were suspended at a concentration
of 300 glomeruli/ml in KRBG medium (1000
glomeruli representing 0.0652 mg of protein; SEM
0.043; n = 13) containing 2.5 mmol/l, 1.5 mmol/l or
no ionized calcium according to the experimental
protocol.
In the first set of experiments, the time course of
basal renin release was studied by incubating 500 pl
of glomerular suspension in five replicates in one of
the three KRBG media for 15, 30, 45 or 60min at
37°C. In a second set of experiments, 5OOpl of
glomerular suspension in one of the three KRBG
media was incubated in six replicates for 60min at
37°C in the presence of lpmol/l or with vehicle
alone. In both sets of experiments, 2Opl of the
supernatants of the samples incubated for different
times were cooled on ice in the presence of 5mmol/l
EDTA and were stored at -20°C until assayed for
renin activity.
Superfusion of dispersed JG cells
An enriched population of JG cells was isolated
from rat kidney cortex and superfused by the
method of Takagi et al. [33] with some modifications. Male Wistar rats (220-260 g), maintained
on a standard diet, were killed by decapitation. The
renal cortices were finely minced into small pieces,
washed in KRBG buffer and incubated with
1mg/ml collagenase (200-400 units/ml) and 1mg/ml
BSA in 25ml of KRBG per kidney at room
temperature for 60min and then at 37°C for
another 30 min. Unless specified, the ionized calcium
concentration and pH of the KRBG were adjusted
to 1.5 mmol/l and 7.4, respectively, as described
above, and the buffer was continuously gassed with
95% 0,/5% CO,. The digested tissue was disag-
13
gregated with a pipette and filtered through two
different sizes of stainless-steel sieves (70 and 25 pm
pore size). These filtrations removed undigested
tissue, glomerulo-vascular and tubular structures,
and cell clumps, and the final filtrate was a separate
cell suspension. This cell suspension was centrifuged
at 80g for 10min and washed twice in KRBG
containing 2mg/l BSA. The cell pellet was reconstituted with lOml of 40% (v/v) iso-osmotic Percoll
per kidney. This suspension was again filtered
through a 50pm sieve to remove the remaining cell
aggregates. Five millimetres of 20% (v/v) iso-osmotic
Percoll solution were gently layered on the same
volume of 40% (v/v) Percoll/cell suspension in a
centrifuge tube. The tube was centrifuged at 300g
for 10min. The cell fraction collected from the
interphase was diluted with about two volumes of
cold KRBG without BSA, centrifuged at 80g for
lOmin, washed twice and reconstituted with 1ml of
the same cold medium containing 2mg/ml BSA per
kidney for renin release studies. Although not pure,
the procedure gave an enriched population of JG
cells. The specific renin activity of the cells determined after freezing and thawing three times was
2.07pg of ANG 1h-Img-I of protein (SEM 0.74;
n = 6), compared with 0.032 pg of ANG I h- mg(SEM 0.007) in the cortex after homogenization in a
Teflon glass tissue grinder and freezing and thawing
three times.
A thermostatically controlled column of 1cm
inner diameter (C10/20 column chromatography;
Pharmacia, Uppsala, Sweden) was prepared by
layering a 1cm height of Sephadex G15 previously
swollen in KRBG medium. The column was washed
thoroughly with KRBG and a 2ml of aliquot of cell
suspension obtained from two kidneys was layered
on to the Sephadex. The column, connected to a
superfusion system and warmed at 37"C, was superfused at a rate of 0.2ml/min using a peristaltic
pump (Minipuls 3; Gilson). The superfusion buffer
was KRBG containing 2mg/ml BSA and various
other substances as specified in the legends of the
Figures and was maintained at 37°C in a water bath
and gassed continuously with 95% 0,/5% CO,. The
column was superfused for an initial 90 min equilibration period, after which 5min fractions of superfusate (1 ml) were collected using a linear fraction
collector (model 201; Gilson). As a general rule the
substances to be tested [( -)-isoprenaline, ( +)verapamil and NlePTH] were added after 29min of
fraction collection to the superfusion medium. The
effect of EGTA in calcium-free medium buffer on
renin release was also studied by changing the
KRBG-BSA (2 mg/ml) medium for a CaC1,-free
KRBG-BSA (2 mg/ml) medium containing 1mmol/l
EGTA at 29min.
Determination of renin activity
Twenty microlitres of the various samples of
perfusate, supernatant or superfusate were incu-
14
C. Saussine et al.
bated, at least in duplicate, with an excess of
partially purified rat substrate (see below), 5 mmol/l
8-hydroxyquinoline (converting enzyme inhibitor)
and 66.6 mmol/l phosphate buffer, pH 6.5 containing
13.5mmol/l EDTA, in a total volume of 1OOpl for
1 h at 37°C. The amount of substrate in each tube
ranged from 200 to 400ng of ANG I equivalent and
the amount of generated ANG I never exceeded
5ng per tube. The reaction was stopped by boiling
for 3min and adding 0.2ml of 66.6mmol/l phosphate buffer, pH 7.5 containing 1 mg/ml sodium
azide and lmg/ml BSA. The suspension was carefully rehomogenized. After centrifugation for 15 rnin
at 2500g, ANG I was measured in 0.1 ml of supernatant by r i a . as described by Fyhrquist et al. [34].
Partially purified angiotensinogen was prepared
by ammonium sulphate fractionation of plasma
obtained from rats bilaterally nephrectomized for
48 h, by the method of Itoh et al. [35]. The various
substrate preparations had neither measurable
ANG I nor renin activity. Angiotensinase activity
was also absent from substrate preparations and
samples since the recovery of added ANG I was
almost 100%.
Renin activities were expressed as ng or pg of
ANG I generated in 1 h of incubation per ml of
perfusate or superfusate. In experiments with isolated glomeruli this activity was expressed as
ANG I h-' (1000 glomeruli)-'. In experiments with
isolated perfused kidney, this activity was normalized per g of kidney weight, taking the weight of
the contralateral kidney as the weight of the perfused kidney (ANG I h-'ml-' g-').
Data points obtained from control and corresponding agonist-treated kidneys, glomeruli or JG
cells were subjected to analysis of variance for
factorial experiments [36] taking the experimental
conditions (with or without agonist) for all levels of
time or concentration as the source of variation.
Where significant differences were found, a Duncan
[37] multiple range and multiple F-test was applied
to identify significant differences between individual
times or concentrations. A 95% confidence level
( P < 0.05) was considered to reflect significant differences. Deviations from the mean values were
expressed as the SEM.
RESULTS
Effect of rPTH-( 1-34) on renin release from isolated
perfused rat kidney
In the first set of experiments we compared the
extent of renin release over 80min of perfusion in
two kinds of independent kidney preparations: the
time-control kidneys and kidneys receiving
125 nmol/l rPTH-( 1-34) at 38 min (designated as
PTH-treated kidneys). The perfusion flows applied
to reach 70mmHg during the first 10-15min were
not significantly different between time-control and
PTH-treated kidneys: 20.4mlmin-' g-' (SEM 2.01;
0
20
40
60
80
Time (min)
Fig. I. Comparison of the timecourse of perfusion pressure ( a ) and
renin release (6) in timecontrol kidneys (0,
n=5) and kidneys
receiving 125nmol/l rPTH-(1-34) at 38min ( 0 , n=4). Values are
means +SEM. Statistical significance: *P <0.05 compared with the timecontrol kidneys for a given perfusion pressure.
n = 5 ) and 22.2mlmin-'g-' (SEM 1.97; n=4), respectively. Since perfusate flows were adjusted during
the first 10-15 min and kept constant throughout
the remainder of the experiment, the perfusion pressures were recorded at 35, 40 (around the time of
the PTH injection in PTH-treated kidneys), 45, 50,
60, 70 and 80min in order to ensure that the
baroreceptors would not be involved in the renin
release responses. Although not statistically significant, PTH decreased the perfusion pressure by
about 3 mmHg at 45 rnin in PTH-treated kidneys as
compared with time-control kidneys (Fig. la). In
both kidney groups, the perfusion increased slowly
and similarly thereafter, reaching 75.9 mmHg (SEM
2.36) in the time-control kidneys and 73.0 mmHg
(SEM 3.62) in the PTH-treated kidneys at 80min.
The renin activity in the recirculating perfusate rose
spontaneously with time in the time-control kidneys
reaching maximal levels between 60 and 70 min
(Fig. Ib). This result is in good agreement with a
previous finding that hyperoncotic perfusate
increased basal renin release from isolated nonfiltering rat kidneys over a period of 70min [38].
Since the rise in perfusion pressure became too high
after 90-100 min, the time-course of renin release
was compared between time-control and PTHtreated kidneys over a 35-80 rnin period, during
which the perfusion pressure changes were limited.
As shown in Fig. l(b), 125nmol/l rPTH-(1-34)
stimulated renin accumulation in the perfusate at all
perfusion times after PTH injection. The maximal
Direct action of parathyroid hormone on renin release in vitro
15
1.4 - ( a )
1.2
-a, 1.0
1
I
al-
-1-F
0.8
el,
c '+
0.6
25
-
-
-
.
'",0.4 -
3
-
0.2
-
0.0
-
level of renin release in PTH-treated kidneys
reached at about 70min was twice as high as that in
time-control kidneys: 1271 (SEM 189) versus 648 ng
of ANG I h-'ml-' g-' (SEM 135).
The second series of experiments was to examine
the concentration-dependent stimulation of renin
release by rPTH-(1-34). As shown in Fig. 2(a), the
renin activity released during the 35 min which
preceeded the PTH injection was similar in timecontrol and PTH-treated kidneys and represented
about 400ng of ANG I h-' ml-' g-I. The administration of rPTH-(1-34) at 38min resulted in a
concentration-dependent increase in renin secretion
over the 35-65min period of perfusion. Presented as
the net renin activity increase from the end of the 035min period (Fig. 2b), rPTH-(1-34) at 1.25, 12.5
and 125nmol/l increased significantly the mean
renin activity from about 300ng of ANG I
h-' ml-' g-' in time-control and 0.0125nmol/l
PTH-treated kidneys to 529, 608 and 787ng of
ANG I h-' ml-' g-', respectively. Due to the cost
of the peptide, a clear-cut plateau was not reached
in these experiments. Therefore, the concentration of
hormone producing half-maximal stimulation of
renin release was not determined.
Effect of NlePTH on renin release from isolated rabbit
glomeruli
Light microscopy revealed that the preparation
consisted of glomeruli without Bowman's capsule.
Occasionally a fragment of an afferent or efferent
arteriole could be seen attached to a glomerulus.
The total renin content of these preparations was
found to be 1.948pg of ANG I h-' (1000
glomeruli)-' (SEM 0.314; n=6).
In the first set of experiments, the time course of
4
-
renin release from isolated glomeruli into the supernatant was studied (Fig. 3 4 . Since the intracellular
calcium concentration is thought to be a signal for
the modulation of renin release, the experiments
were performed to determine whether changes in the
ionized calcium concentration of the KRBG would
affect basal or NlePTH-induced stimulation of renin
release. Renin release increased linearly with time up
to 60min regardless of the calcium concentration in
the incubation medium. Increasing the calcium
concentration from 1.5 to 2.5 mmol/l slightly, but
significantly, decreased the slope of the regression
line (renin release rate), whereas omitting calcium
from the KRBG markedly increased the rate of
renin release.
In the second set of experiments, the effect of
1 pmol/l NlePTH on the renin activity released
during 60min of incubation was studied. In KRBG
medium containing 1.5 mmol/l calcium, NlePTH
consistently produced an approximate 55% increase
in supernatant renin activity. Basal renin activity
was 67.2 (SEM 7.25) and increased significantly to
104ng of ANG I h - ' (1000 glomeruli)-' (SEM 10.9;
n = 4) in response to NlePTH. Omitting the addition
of calcium significantly augmented the basal renin
activity in the supernatant from 67.2 (SEM 7.25) to
21 1 ng of ANG I h- (1000 glomeruli)-' (SEM
14.6), but completely abolished the effect of NlePTH
on renin release: 211 (SEM 14.6) versus 188ng of
ANG I h-' (1000 glomeruli)-' (SEM 15.1). Increasing the calcium concentration in the KRBG
medium to 2.5 mmol/l significantly reduced basal
activity from 67.2 (SEM 7.25) to 45.0ng of
ANG I h - ' (1000 glomeruli)-' (SEM 5.74) and
NlePTH effectively stimulated renin release by an
approximately 128% increase in supernatant renin
activity: 45.2 (SEM 5.74) versus 103ng of
'
C. Saussine et al.
16
~
I pmol/l NlePTH
nn
r
I
I
I
I
I
0
15
30
45
60
1
*
I
*
I
2.5
I.5
None
Ca2+ concn. (mmol/l)
Time (MIN)
Fig. 3. Renin release by isolated rabbit glomeruli. (a) Effect of changing the calcium concentration of KRBG on the timecourse
of renin release from isolated rabbit glomeruli into the supernatant. Each data point is the mean+SEM of three independent
experiments in which renin activities were measured in five replicates. (b) Effect of I pmol/l NlePTH on renin release from isolated
rabbit glomeruli incubated for 60min in calcium-free KRBG or in KRBG containing 1.5 or 2.5mmol/l calcium. Each bar is the mean of
four independent experiments in which renin activities were determined in six replicates. Statistical significance: *P <0.05.
Abbreviation: NS, not significant. Values are means SEM.
+
ANG I h - ' (1000 glomeruli)-' (SEM 9.8). In addition, the net magnitude of the response to NlePTH
was higher than that obtained with 1.5mmol/l
calcium.
Effect of NlePTH on renin release from superfused
dispersed cortical cells enriched in JG cells
In order to ensure that these superfused
collagenase-dispersed cortical cells enriched in JG
cells were responsive to known stimulators of renin
release, verapamil, isoprenaline and EGTA were
used as promoters of renin release. Fig. 4 shows
typical data obtained from four independent experiments. After 29min of superfusion with KRBG
buffer containing 2 mg/ml BSA, cells were subjected
to 0.25 mmol/l verapamil, 100 nmol isoprenaline or
calcium-free KRBG-BSA (2 mg/ml) containing
lmmol/l EGTA. It is well known that ascorbic acid
prevents the oxidation of isoprenaline. We also
observed in preliminary experiments that the stimulatory responses to verapamil in the absence of
ascorbic acid were low and short-lived. Therefore
0.1 mmol/l ascorbic acid was added together with
verapamil or isoprenaline at 29 min. Both compounds, as well as EGTA, induced sustained stimulation of renin release as compared with the basal
renin activity in the seven fractions (5min each)
immediately before the onset of the stimulation of
renin release (Figs. 4b-44. In synchronously run
control columns (Fig. 4a), which received vehicle
alone at 29min, renin release was stable over a
period of 100min of perfusion.
Expressed as the percentage of the average basal
renin activity of the seven fractions immediately
before the onset of the stimulation of renin release,
5
1
I
0
I
50
75
Superfusion time (min)
25
100
Fig 4. Representative data from four independent experiments illustrating the effects of 0.1 mmol/l ascorbic acid alone (a, timecontrol) or with 0.25mmol/l verapamil (b), 100 nrnol/l isoprenaline
(c) and I mmol/l EGTA in calcium-free medium ( d ) on renin
release from superfused dispersed JG cells isolated from rat renal
cortex. The horizontal broken line indicates the time of drug addition
(29 min).
Direct action of parathyroid hormone on renin release in vitro
I
/i
I
1
T
0
0
, ,o.o,o‘ 00-0-0
0
O.O’O
30
I
0
,
I
20
.
I
40
,
I
10
20
30
40
Superfusion time (min)
50
60
Fig. 6. Mean effect of IWnmol/l NlePTH on renin release from
collagenaredispersed rat kidney cortex cells enriched in JG cells
calculated from the data presented in Fig. 5 and expressed as the
percentage of the average basal renin activity of the seven fractions
(0-35 min) immediately before the onset of renin stimulation. Statistical significance: *P <0.05 compared with basal renin activity. Values are
meansf SEM (n=4).
\o/o’
D\
17
.
60
Superfusion time (min)
Fig. 5. Individual data illustrating the timecourse of renin release
from superfused JG cells in response to IWnmol/ NlePTH (a)
added at 29min (-----) and in time control experiments (4.
In
these experiments the superfusate was KRBG buffer containing 2mg/ml
BSA and 0.3 mmol/l PMSF.
cumulated data from these experiments showed that
verapamil, isoprenaline and EGTA significantly
increased renin release by 155.3% (SEM 13.4; n=4),
200.2% (SEM 11.3; n=4) and 230.0% (SEM 17.0;
n = 5), respectively. These results demonstrate that
these cell preparations were able to display sustained changes in renin release in response to
agonist action.
We then explored, under the same conditions, the
effect of lOOnmol/l NlePTH on the renin release
from these cells. In these experiments 0.3 mmol/l
phenylmethanesulphonyl fluoride (PMSF), a protease inhibitor, was added together with ascorbic
acid. In preliminary experiments, we observed that
PMSF by itself exhibited stimulating effects on
renin release and that NlePTH was unable to
stimulate renin release in the absence of PMSF.
Therefore both PMSF and ascorbic acid were added
from the onset of superfusion. Fig. 5 shows the
individual data obtained from six independent
experiments. NlePTH effectively stimulated renin
release in each experiment as compared with basal
renin activity (Fig. 5 4 and with time-control columns (Fig. 5b). The increase in renin release began
between 5 and 10min after NlePTH addition and
was short-lasting, as the renin activity returned to
baseline value within 25 min. Cumulated data from
these experiments (Fig. 6), expressed as the percentage of the average renin activity of the seven
fractions immediately before the onset of renin
stimulation, showed that the maximal response
represented 147.1% (SEM 7.2; n=4) of the basal
renin activity.
DISCUSSION
In a previous study in isolated rat kidney preparations, we suggested that PTH could directly
stimulate renin release [30]. In order to minimize
the effects of haemodynamic changes and tubular
transport on the release of renin and to avoid PTH
degradation at tubular sites, these studies were
performed under non-filtering conditions [32]. In
the present studies, we have extended these initial
observations by examining two other aspects of
PTH action on renin release: (i) the PTH
concentration-related responses of renin release in
the same model of the isolated rat kidney; (ii) the
direct stimulation by PTH of renin release from
isolated glomeruli or JG cells. The present findings
clearly show that PTH can directly stimulate renin
release in the three different preparations in vitro.
In the non-filtering rat kidney preparations, the
low perfusion pressure and the absence of preexisting vascular tone suppressed the renal vasodilating effect of PTH previously described by this
laboratory [121. Since the perfusion pressures were
maintained within a narrow range (less than
6 mmHg), variations in renin release in response to
PTH would be expected to originate from JG renin-
18
C. Saussine et al.
secreting cells and not from an alteration in the
stability of the kidney preparations. Under these
conditions, rPTH-( 1-34) at 125 nmol/l consistently
stimulated renin release as compared with timecontrol kidneys, in good agreement with our previous findings [30]. In the present work we have
also shown that PTH stimulated renin release from
isolated non-filtering rat kidney in a concentrationrelated manner with a threshold concentration of
1 nmol/l.
Isolated glomeruli or JG cells have been generally
used to explore the physiological and biochemical
aspects of renin release in response to a variety of
biological agents [22, 23, 39, 401. These approaches
are thought to have several physiological and technical advantages over the recirculating kidney preparation. They reduce the indirect effects of PTH on
renin release. In addition, superfused dispersed cells
can be continuously exposed to fresh buffer and,
hence, secretory or metabolic products which interfere with renin secretion are not allowed to accumulate. Moreover, each cell or glomerular preparation
can serve as its own control.
The present study also examined the effects of
NlePTH, another well-known bioactive PTH fragment [41], on renin release from isolated glomeruli,
particularly during extracellular calcium buffering.
Increasing the extracellular calcium concentration
inhibited basal renin release. Conversely, a lowcalcium medium increased markedly basal renin
release. This is in agreement with a number of
previous reports [22, 23, 42, 431.
The stimulating properties of NlePTH were displayed only when extracellular ionized calcium was
present and even more as the extracellular calcium
concentration increased. These results suggest that
an inhibition of calcium influx is involved in the
action of NlePTH on renin release. This is further
supported by our previous study in the isolated
perfused kidney, where PTH was ineffective in the
presence of verapamil [30]. Conversely, the drastic
inhibitory action of the calcium-channel opener
BAY-K8644 on basal renin release was reversed
when PTH was added to the perfusion medium
[30]. In recent reports, Pang and co-workers [20,
211 proposed that PTH could be the first identified
endogenous circulating hormone capable of inhibiting voltage- and time-dependent calcium channels in
vascular smooth muscle cells. Since renin-secreting
JG cells are modified smooth muscle cells, these
results further support the view that PTH stimulates
renin release through inhibition of the influx of
extracellular calcium in addition to the increase in
intracellular cyclic AMP concentration. Direct
measurements of the cytosolic calcium concentration
of the renin-secreting cells in response to PTH will
be required to resolve this point definitely.
In the last part of this study, we have investigated
whether the effect of NlePTH can be demonstrated
directly in isolated JG cells. Although these cellular
preparations may not be pure, they were able to
readily display changes in renin release in response
to known renin promoters such as isoprenaline,
verapamil and EGTA. Under similar conditions,
NlePTH repeatedly stimulated basal renin release
but unlike isoprenaline, verapamil and EGTA, these
changes were short-lived. In this regard, it is interesting to compare the isolated J G cells with the
isolated perfused rat kidney. In the former study,
renin release returned to the baseline level within
25min, whereas in the latter, the increase in renin
release stopped 35-40min after PTH injection. The
half-life of PTH in the isolated perfused kidney may
be longer than in isolated JG cells in which PTH
may be catabolized by tubular cells probably still
present in this preparation. Indeed, NlePTH was
unable to stimulate renin release from JG cells in
the absence of PMSF. An additional explanation is
that both preparations were rapidly desensitized to
PTH when continuously exposed to the hormone.
This suggestion is consistent with our previous
findings that sustained exposure of the renal vascular bed led to resistance or tachyphylaxis [12].
An increased plasma renin activity has been
reported in some hypertensive patients with primary
hyperparathyroidism, but not in normotensive
patients and those with secondary hyperparathyroidism [28, 44, 451. The high circulating level of
PTH in some hypertensive patients with primary
hyperparathyroidism [46] could stimulate renin
release, favouring an elevation in blood pressure.
However, the present study does not support such
an hypothesis since the lowest PTH concentration
inducing a significant increase in renin release was
1 nmol/l (Fig. 2). Although PTH exhibits significant
actions on the renal vascular bed, including renal
vasodilatation [121, occupancy of preglomerular
receptors [161 and stimulation of adenylate cyclase
[ 13-15], and direct renin stimulating actions [30]
(and the present study), the question of whether
PTH has a physiological role in the regulation of
the renal microcirculation remains unresolved. However, some evidence supports a role for PTH in the
regulation of renal blood flow. Thus an association
has been shown between delayed renal allograft
functions and low concentrations of circulating
PTH in recipients at the time of transplantation
[47]. It has also been noted that PTH has a renal
vasodilator effect in cyclosporin-treated rats [47].
In summary, PTH significantly stimulated renin
release in the isolated perfused kidney, isolated
glomeruli and isolated J G cells. The results obtained
in isolated glomeruli and JG cells further support a
direct stimulatory action of PTH on renin release.
Moreover, PTH action on renin release is
concentration-related in the isolated kidney and is
dependent upon extracellular calcium as shown in
the isolated glomeruli, The modulation of PTHinduced renin release by calcium is compatible with
the reported ability of PTH to block calcium channels and relax vascular smooth muscle cells.
Direct action of parathyroid hormone
ACKNOWLEDGMENTS
We thank Professors C. Bollack and J. Geisert
(CHRU, Strasbourg) for advice and encouragement.
We also thank Ms J. Krill and S. Rothhut for their
expert technical assistance and competent secretarial
support. This work was supported by INSERM
(National Institute of Health and Medical Research)
(grant CRE 897005), ARC (Cancer Research
Association) (grant 6104) and endowments of the
UFR of Medicine, Louis Pasteur University,
Strasbourg.
REFERENCES
I. Berthelot A, Gairard A. Action of parathormone on arterial blood pressure
and on contraction of isolated aorta in the rat. Experientia 1975; 31: 457-8.
2. Schleiffer R, Berthelot A, Gairard A. Action of parathyroid extract on arterial
blood pressure and on contraction and ‘ICa exchange in isolated aorta of the
rat. Eur J Pharmacol 1979; 58: 163-7.
3. Driessens M, Vanhoutte PM. Effect of calcitonin, hydrocortisone, and
parathyroid hormone on canine bone blood vessels. Am J Physiol 1981; 241:
H91-94.
4. Rambausek ME, Ritz W, Rascher W, Kreusser W, Mann JFE, Kreye VA, et al.
Vascular effects of parathyroid hormone (PTH). Adv Exp Med Biol 1982; 151:
619-32.
5. Suzuki Y, Lederis K, Huang M, LeBlanc FE, Rorstad OP. Relaxation of bovine,
porcine and human brain arteries by parathyroid hormone. Life Sci 1983;3 3
2497-503.
6. Nickols GA. Increased cyclic AMP in cultured vascular smooth muscle cells
and relaxation of aortic strips by parathyroid hormone. Eur J Pharmacol 1985;
1 1 6 1374.
7. Pang PKT, Yang MCM, Shew R, Tenner TE, Jr.The vasorelaxant action of
parathyroid hormone fragments on isolated rat tail artery. Blood Vessels 1985;
22: 57-64.
8., Nickols GA, Metz MA, Cline WH, Jr.Vasodilation of the rat mesenteric
vasculature by parathyroid hormone. J Pharmacol Exp Ther 1986; 236: 419-23.
9, Crass MF, Ill,Jayaseelan CL, Darter TC. Effects of parathyroid hormone on
blood flow in different regional circulations. Am J Physiol 1987; 253 R6349.
10. Wang H-H, Drugge ED, Yen YC, Blumenthal MR, Pang PKT. Effects of
synthetic parathyroid hormone on hernodynamics and regional blood flows.
Eur J Pharmacol 1984; 97: 209-15.
I I. Pang PKT, Janssen HF, Yee ]A. Effects of synthetic parathyroid hormone on
vascular beds of dogs. Pharmacology 1980 21: 213-22.
12. Musso MJ, Barthelmebs M, lmbs JL, Plante M, Bollack C, Helwig 11. The
vasodilator action of parathyroid hormone fragments on isolated perfused rat
kidney. Naunyn-Schmiedeberg’s Arch Pharmacol 1989; 340: 246-51.
13. Helwig JJ, Schleiffer R, Judes C, Gairard A. Distribution of parathyroid
hormonesensitive adenylate cyclase in isolated rabbit renal cortex microvessels and glomeruli. Life Sci 1984; 3 5 2649-57.
14. Helwig JJ, Yang MCM, Bollack C, Judes C, Pang PKT. Structure-activity
relationship of parathyroid hormone: relative sensitivity of rabbit renal
microvessel and tubule adenylate cyclases t o oxidized PTH and PTH
inhibitors. Eur J Pharmacol 1987; 140: 247-57.
15. Musso MJ, Plante M, Judes C, Barthelmebs M, Helwig JJ.Renal vasodilation
and microvessel adenylate cyclase stimulation by synthetic parathyroid
hormondike protein fragments. Eur J Pharmacol 1989; 174: 139-51.
16. Nickols GA, Nickols MA, Helwig JJ. Binding of parathyroid hormone and
parathyroid hormonerelated protein to vascular smooth muscle of rabbit
renal microvessels. Endocrinology (Baltimore) 1990; I=. 721-7.
17. Barajas L. Anatomy of the juxtaglomerular apparatus. Am J Physiol 1979 236
F333-43.
18. Taugner R, Biihrle CP, Hackenthal E, Mauneck E, Nobiling R. Morphology of
the juxtaglomerular apparatus and secretory mechanisms. Contrib Nephrol
1984; 43: 76401.
19. Cantin M. Aranjo-Nascimento MF, Benchimol S, Desormeaux Y. Metaplasia of
smooth muscle cells into juxtaglomerular cells in the juxtaglomerular
on
renin release in
vitro
19
apparatus, arteries and arterioles of the ischemic (endocrine) kidney. Am J
Pathol 1977; 87: 581-602.
20. Pang PKT. Wang R, Shan J, Karpinski E, Benishin CG. Specific inhibition of
long-lasting, L-type calcium channels by synthetic parathyroid hormone. Proc
Natl Acad Sci USA 1990 87: 623-7.
21. Wang R, Karpinski E, Pang PKT. Parathyroid hormone selectively inhibits
L-type calcium channels in single vascular smooth muscle cells of the rat.
J Physiol (London) 1991; 441: 325-46.
22. Fray JCS, Park CS, Valentine AND. Calcium and the control of redin
secretion. Endocr Rev 1987; 8: 53-93.
23. Hackenthal E, Paul M, Ganten D, Taugner R. Morphology, physiology, and
molecular biology of renin secretion. Physiol Rev 1990; 7 0 1067-1 16.
24. McCredie DA, Powell HR, Rotenberg E. Effect of parathyroid extract on
renin release in the dog. Clin Sci Mol Med 1975;48: 461-3.
25. Powell HR, McCredie DA, Rotenberg E. Renin release by parathyroid
hormone in the dog. Endocrinology (Baltimore) 1978; 103 985-9.
26. Smith JM, Mouw DR, Vander A]. Effect of parathyroid hormone on plasma
renin activity and sodium excretion, Am J Physiol 1979; 236 F3Il-19.
27. Keeton TK, Campbell WB. The pharmacologic alteration of renin release.
Pharmacol Rev 1981; 31: 81-227.
28. Horky K, Broulik P, Pacovsky V. The effect of parathyroid hormone on
plasma renin activity in humans and hypertension in patients with primary
hyperparathyroidism. J Hypertens 1986; 4 S585-7.
29. Agur ZS,Gardner LB, Beck LH, Goldberg M. Effects of parathyroid hormone
on renal tubular reabsorption of calcium, sodium, and phosphate. Am J
Physiol 1973; 224 1143-8.
30. Helwig JJ, Musso MI, Judes C, Nickols GA. PTH and calcium: interactions in
the control of renin secretion in the isolated nonfiltering rat kidney.
Endocrinology (Baltimore) 1991; 129 1233-42.
31. Schurek HI, Brecht JP, Lohfert H, Hierholzer K. The basic requirements for
the function of the isolated cell free perfused rat kidney. Pfliigers Arch 1975;
354 349-65.
32. Maack T. Physiological evaluation of the isolated perfused rat kidney. Am J
Physiol 1980 2% F71-8.
33. Takagi M, Takagi M, Franco-Saenn R, Mulrow PJ. Effect of atrial natriuretic
peptide on renin release in a superfusion system of kidney slices and
dispersed juxtaglomerular cells. Endocrinology (Baltimore) 1988; In 1437-42.
34. Fyhrquist F, Soveri P, Puutula L, Stenman UH. Radioimmunoassay of plasma
renin activity. Clin Chem 1976; 22: 2504.
35. ltoh S, Carretero OA, Murray RD. Renin release from isolated afferent
arterioles. Kidney Int 1985;27: 762-7.
36. Winer BJ. Design and analysis of factorial experiments. Statistical principles in
experimental design. New York: McGraw-Hill, 1971.
37. Duncan DB. Multiple range and multiple F tests. Biometrics 1955; II: 1-41.
38. Cohen AJ, Spokes K, Brown RS, Stoff IS, Silva P. Stimulation of renin release
by hyperoncotic perfusion of the isolated rat kidney. Circ Res 1982; 5 0
400-4.
39. Fray JCS. Control of renin secretion by extracellular calcium. Cell Calcium
1990 II: 339-41.
40. Fray JCS. Regulation of renin secretion by calcium and chemiosmotic
forces-(patho)physiological
considerations. Biochim Biophys Acta 1991; IOW:
243-62.
41. Segre GV, Rosenblatt M, Reiner BL, Mahaffey JE, Potts IT, Jr. Characterization
of parathyroid hormone receptors in canine renal cortical plasma membranes
using a radioiodinated sulfur-free hormone analogue. Correlation of binding
with adenylate cyclase activity. J Biol Chem 1979; 254 69804.
42. Pardy K, Williams BC, Noble AR. Inhibitory role of Caz+ in the control of
renin secretion: a study using superfused dispersed rat renal cortical cells.
Clin Sci 1989; 77: 273-9.
43. Nisbet )A. Comparison of three parathyroid hormone assays. Ann Clin
Biochem 1986; 23: 429-33.
44. Dominiczak AF, Lyall F, Morton JJ, et al. Blood pressure, left ventricular mass
and intracellular calcium in primary hyperparathyroidism. Clin Sci 1990; 78:
127-32.
45. Brinton GS, Jubiz W, Lagerquist LD. Hypertension in primary
hyperparathyroidism: the role of the renin-angiotensin system. J Clin
Endocrinol Metab 1975;41: 1025-9.
46. Hellstrom J, Brike G, Edvall GA. Hypertension in hyperparathyroidism. Br J
Urol 1958; 30: 13-24.
47. Ferguson CJ, Williams ID, Silver A, Woodhead IS, Salaman JR. Effects of
parathyroid hormone on delayed renal allograft function. Br. Med J 1991; 303
287-8.