Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026
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ABSTRACT |
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It has been well documented
that low concentrations of ANG II (1011 to
10
10 M) stimulate, whereas high concentrations of ANG II
(10
8 to 10
5 M) inhibit Na+
transport in proximal tubules of rat and rabbit kidneys. Measured ANG
II concentration in proximal tubular fluid is in the nanomolar range.
In the present study, we investigated the role of PKC, intracellular
Ca2+, and cAMP in modulating the effects of luminal ANG II
on Na+ absorption by microperfusion techniques in rabbit
superficial segment of proximal tubules in vitro. We confirmed that ANG
II (10
9 M) had no change on fluid absorption
(Jv); however, fluid absorption increased
significantly when 10
9 M ANG II and
3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8), a
blocker of intracellular calcium mobilization, were added together. In
contrast, ANG II significantly decreased Jv when
PKC was inhibited. When 10
9 M ANG II was present together
with 1-(5-isoquinolinesulfonyl)-2-mehtylpiperazine and TMB-8, no
significant change of Jv occurred. Inhibition of endogenous cAMP activity by a PKA inhibitor did not change either basal
or ANG II-stimulated fluid absorption. Our results indicate that ANG II
regulates Na+ absorption by a cAMP-independent mechanism
and that PKC and intracellular calcium both play a critical role in
modulating the effects of physiological concentration of ANG II on
proximal tubule transport. Balance between these two cytosolic
messengers modulates the effects of ANG II on fluid absorption in the
proximal tubule.
Na+ transport; proximal tubule; microperfusion
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INTRODUCTION |
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ANG II IS AN IMPORTANT
HORMONAL regulator of Na+ and HCO11 to 10
10
M) stimulate, whereas high concentrations (10
8 to
10
5 M) inhibit Na+ transport in both rats and
rabbits (6, 11, 20, 21). Blocking ANG II receptors
systemically decreases Na+ and HCO
8 to
10
6 M) of ANG II increase cell Ca2+ and that
nanomolar concentrations of ANG II increase PKC activity and
intracellular Ca2+ mobilization in the primary cultured
rabbit renal proximal tubule cells (10). In the present
study, we examined the functional role of cAMP, PKC, and intracellular
Ca2+ in modulating the effect of ANG II on Na+
absorption in proximal tubules of rabbit kidney in vitro. Our results
show that Na+ transport is not inhibited by endogenous
cAMP, whereas both PKC and cytosolic Ca2+ modulate the
effects of the nanomolar concentration of ANG II. Dual effects of ANG
II of either stimulation or inhibition of transport may be the result
of different levels of Ca2+ and PKC activation in proximal
tubule cells.
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MATERIALS AND METHODS |
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Superficial proximal convoluted tubules (S2 segments) were dissected and perfused in vitro by using conventional methods (9). Briefly, kidneys from adult female New Zealand white rabbits weighing 2-3 kg were removed and cut into coronal slices. Individual tubules were dissected in cooled (4°C) Hanks' solution containing (in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 1 MgCl2, 10 Tris · HCl, 0.25 CaCl2, 2 glutamine, and 2 L-lactic acid. The solution was bubbled with 100% O2 and had a pH of 7.4.
S2 segments of the proximal tubules were perfused with an ultrafiltrate-like solution containing (in mM) 125 NaCl, 22 NaHCO3, 1 CaCl2, 1.2 MgSO4, 2 glutamine, 2 lactic acid, 10.5 glucose, 5 KCl, and 1.2 phosphoric acid. A similar solution containing (in mM) 101 NaCl, 22 NaHCO3, 1 CaCl2, 1.2 MgSO4, 2 glutamine, 2 lactic acid, 10.5 glucose, 5 KCl, 1.2 phosphoric acid, and 32.5 HEPES was used as bath medium. Perfusate and bath solutions were bubbled with 95% O2-5% CO2 and had a pH of 7.4. Osmolalities of the bath and perfusate were adjusted to 300 mosmol/kgH2O by the addition of either H2O or NaCl. The bath solution also contained 3 g/dl albumin. Bath fluid was continuously changed at a rate of at least 0.5 ml/min to maintain the constancy of pH and bath osmolality.
ANG II and several inhibitors were added to the luminal solutions. All tubules were perfused at a rate of 10-20 nl/min at 37-38°C in a 1.2-ml temperature-controlled bath. The first period of collection began after an equilibration time of 30-60 min. Net fluid volume absorption (Jv) was measured with [methoxy-3H]inulin. The extensively dialyzed (methoxy-3H)-inulin was added to the perfusate at a concentration of 30 µCi/ml as a volume marker. For each experimental period, three timed collections of tubular fluid were made, the volume of the perfusate and collected samples were measured in a constant-bore glass capillary, and [3H]inulin concentrations in those samples were determined in a liquid scintillation counter (model LS5801; Beckman).
The rate of net fluid reabsorption was calculated according to the
following equation: Jv = Vo VL, where
VL is the measured rate of fluid collection and
Vo = VL (INL/INo). INL/INo is
the ratio of radioactive inulin of collected and original perfusion
fluid. The rates of fluid absorption were expressed per millimeter of
the proximal tubule.
ANG II was purchased from Peninsula Laboratories (Belmont, CA), and [3H]inulin was purchased from DuPont-New England Nuclear (Boston, MA). KT-5720 was purchased from Calbiochem (La Jolla, CA) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8), 1-(5-isoquinolinesulfonyl)-2-mehtylpiperazine (H-7), and all other chemicals were obtained from Sigma (St. Louis, MO).
All data were presented as means ± SE. Student's t-test was used to compare control and experimental groups. The ANOVA test was used for comparison of several experimental groups with a control group. The difference between the mean values of an experimental group and a control group was considered significant if P < 0.05.
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RESULTS |
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Effect of luminal ANG II on fluid absorption. In the first series of experiments, we confirmed that a low concentration of ANG II stimulates fluid absorption in the S2 segment of the proximal tubule in vitro, as previously observed in the rat in vivo (12, 21). Proximal tubules were microperfused with an ultrafiltrate-like solution containing 125 mM NaCl and 22 mM NaHCO3. Under these conditions, volume reabsorption (Jv) results predominantly from net Na+ absorption.
As shown in Table 1 and Fig. 1, the addition of 10
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Effect of PKA inhibitor and ANG II on proximal tubule fluid
absorption.
On the basis of the observation that ANG II decreases cAMP
production and stimulates Na+/H+ exchange
(NHE), it was reported that ANG II increases the Na+ and
HCO1 · mm
1).
A similar concentration has been reported by other laboratories (8, 16) to inhibit cAMP in proximal tubule cells. This
result indicates the net Na+ absorption is not inhibited by
endogenous cAMP in the proximal tubule. Additional data shown in Fig. 2
and Table 2 are the effects of both KT-5720 and ANG II on fluid
absorption. Two concentrations of ANG II 10
11 M and
10
9 M were examined. Jv was
significantly increased (0.69 ± 0.06 nl/min) when ANG II
10
11 M and KT-5720 were added together and was 0.32 ± 0.02 nl · min
1 · mm
1
when ANG II 10
9 M and KT-5720 were added together. There
were no significant differences between ANG II alone and ANG II plus
PKA inhibitor. This result indicates that ANG II stimulates
Na+ absorption in the proximal tubule by cAMP-independent
mechanisms.
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Effect of TMB-8 and H-7 on PCT fluid absorption.
Because stimulation of proximal NHE by PKC has been demonstrated
(25), we investigated whether this kinase modulates the effects of ANG II at concentrations that do not alter fluid and Na/HCO9) or of H-7 alone did not change
Jv. However, Jv decreased
significantly when both H-7 and ANG II (10
9) were present
in the perfusion solutions. These data suggest that ANG II stimulates
Jv through PKC activation.
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DISCUSSION |
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In this study, we first confirmed previous observations that ANG
II stimulates Na+ absorption at a concentration of
1011 M but not at 10
9 M in the proximal
tubule (21). Second, we have shown that inhibition of
endogenous cAMP activity by blocking of PKA did not increase Na+ absorption. Third, we have demonstrated that ANG II at
a concentration of 10
9 M produces stimulation or
inhibition of Na+ transport, depending on whether cytosolic
Ca2+ mobilization or PKC activity is inhibited. Our data
are consistent with the conclusion that both PKC and cytosolic
Ca2+ modulate the effects of nanomolar concentration of ANG
II. These results also suggested that the luminal effects of ANG II are modulated by second messengers, such as Ca2+ and PKC.
Because the physiological concentration of ANG II in tubule fluid is
within the nanomolar range, the modulation of the effects of ANG II may
be important for regulating absorption of Na+ in proximal
tubules. It should be noted that under our experimental condition, ANG
II was only added to the luminal side. It is not clear whether the
addition of different concentrations of ANG II in the bath will
modulate luminal ANG II effects.
Previous studies (11, 19, 20) in rat and rabbit kidneys
had shown that ANG II has a biphasic effect on Na+ and
HCO12 to 10
11 M) stimulate and high doses
of ANG II (10
8 to 10
5 M) inhibit
HCO
11 M, whereas maximal inhibition
was observed at 10
6 M (21). Similar results
were also reported by Liu and Cogan (14) by using
microperfusion of early proximal tubule in vivo. With the use of in
vitro microperfusion of rabbit proximal tubule, it was reported that
the effects of ANG II differed, depending on the site of application;
maximal stimulation resulted from application of 10
11 M
in the lumen and 10
10 M induced maximal stimulation on
the basolateral side (12). However, the effects of
nanomolar concentrations of ANG II in modulating proximal tubule
transport have not been explored.
It was demonstrated (5, 26) that cAMP inhibits NHE and
decreases Na+ and HCO11 M and KT-5720 significantly increased
Jv compared with both control and KT-5720
groups. The addition of ANG II 10
9 M and KT-5720 did not
change Jv, which was similar to ANG II 10
9 M alone. These results indicate that ANG II modulates
proximal tubule transport independently in cAMP and the PKA signaling pathway.
Experimental data (6, 24, 25) indicate that both
activation of PKC and intracellular Ca2+ modulate the
proximal tubule transport of Na+ and HCO8 to 10
6 M) of ANG
II increased cell Ca2+ and reduced Na+
absorption in rabbit proximal tubule (6, 10). It has also been shown that ANG II activates the phospholipase C signaling pathway
(27); thus increased release of both
inositol-1,4,5,trisphosphate [Ins(1,4,5)IP3]
and diacylglycerol elevates cytosolic Ca2+ and active PKC,
respectively. Experimental data (10, 23) show that PKC
inhibitors abolish the elevation of fluid and HCO
9 ANG II alone is
consistent with the documented additional action of ANG II of
increasing Ca2+ mobilization. The latter inhibits
transport and thus opposes stimulation by PKC.
Nanomolar concentrations of ANG II increase
Ins(1,4,5)IP3 (EC50 = 2.9 nM)
and intracellular Ca (EC50 = 5.5 nM) in the proximal tubule (18). This suggests that an increase in cell
Ca2+ is the cellular mechanism of ANG II inhibition.
Previous studies (10, 23) have also demonstrated that ANG
II >10 nM increases cell Ca2+ and reduces Na+
and HCO9 M) increases calcium
mobilization and decreases proximal tubule transport. Our studies show
that opposite effects, simultaneous stimulation and inhibition of
tubule transport by activation of PKC and by increased Ca2+
mobilization, cancel each other, so that ANG II has no net effect on
Jv. Our results support the view that ANG II
activation of PKC and increased cell Ca2+ are major
mechanisms mediating its biphasic effects on proximal tubule
Na+ transport.
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ACKNOWLEDGEMENTS |
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We thank Drs. Gerhard Giebisch, Steven Hebert, and Yuehan Zhou for providing assistance and constructive comments.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-17433.
Portions of this study were previously published in abstract form (7).
Address for reprint requests and other correspondence: T. Wang, Dept. of Cellular and Molecular Physiology, Yale School of Medicine, 333 Cedar St., P.O. Box 208026, New Haven, CT 06520-8026 (E-mail: tong.wang{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 14, 2003;10.1152/ajprenal.00261.2002
Received 19 July 2002; accepted in final form 6 December 2002.
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