1 Institut National de la Santé et de la Recherche Médicale Unité 356, Université Paris VI, 3 Hôpital Broussais, Assistance Publique, 75270 Paris Cédex 06, France; and 2 Departments of Medicine and Physiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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The present study was designed to determine the
Na/H exchanger isoforms present in luminal membrane
vesicles (LMV) isolated from rat kidney cortical tubule suspensions, as
well as the effects of acute phorbol ester (phorbol myristate acetate,
PMA) and angiotensin II (ANG II) pretreatment of suspensions on NHE
activity and protein kinase C (PKC) isoform abundance. In LMV, both
NHE3 and NHE2 proteins were found by Western blot analysis, but only
ethylisopropylamiloride-sensitive and almost completely
Hoe-694-resistant Na/H exchange activity was observed from
22Na uptake and thus attributed to
NHE3. PMA pretreatment increased Na/H exchange activity and PKC
isoforms ,
, and
abundance in LMV, and these effects were
prevented by PKC inhibition. Low-dose ANG II
(10
11 M) pretreatment
increased Na/H exchange activity and only PKC-
abundance in LMV, and
these effects were also prevented by PKC inhibition. After high-dose
ANG II (10
7 M), Na/H
exchange activity was decreased in LMV. PKC inhibition did not prevent
this effect. In conclusion, the stimulating effects of PMA and low-dose
ANG II are explained by the translocation of different isoforms of PKC
in LMV, whereas the inhibitory effect of high-dose ANG II is not PKC dependent.
sodium/hydrogen exchanger; protein kinase C; angiotensin II; phorbol esters
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INTRODUCTION |
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FOUR ISOFORMS of Na/H antiporters have been described
so far in the kidney and designated as NHE1 to NHE4. NHE1 and NHE4 are located in the basolateral membrane of the renal tubule (6, 8), and
NHE3 and NHE2 are apical isoforms (5, 9). NHE3 has been detected by
Western blot analysis in kidney cortex brush-border membranes (1, 5)
and by immunohistochemistry in apical membranes of rat and rabbit
proximal tubule (1, 7). Phorbol esters, which stimulate protein kinase
C (PKC), inhibit NHE3 in cultured cells, proximal tubular-like cell
lines originating from kidney tissue, and NHE-deficient fibroblasts
transfected with NHE3 (15, 18, 20). However, previous
results have demonstrated that direct activation by phorbol esters of
endogenous PKC present in luminal membrane vesicles (LMV) isolated from
rabbit kidney cortex increases the Na/H exchange (27). In addition,
phorbol esters acutely applied either basolaterally or luminally
stimulate the luminal Na/H activity in rat proximal tubule perfused in
vivo (19, 25). However, an inhibitory effect was also observed in
proximal tubule after prolonged application of phorbol esters (3, 25).
Several explanations have been proposed for the opposed effects of
phorbol esters in the proximal tubule and in cultured cells. First, the
increase in apical Na/H exchange activity in the proximal tubule in
response to phorbol esters may be due to the activation of an
additional apical NHE isoform, NHE2. However, an increasing body of
evidence supports that NHE2 is not present in the proximal tubule. We
have not found NHE2 protein in the rat proximal tubule by
immunocytochemical method using polyclonal anti-NHE2 antibody (9). NHE2
mRNA has not been detected in rat proximal tubule by in situ
hybridization approach (12). Hoechst 694 (Hoe-694, 100 µM), which
completely inhibits NHE2 transport activity and only marginally NHE3
activity, has no effect on HCO3
absorption in the proximal tubule perfused in vivo (26). Finally, in
mice lacking NHE3, the degree of inhibition of
HCO
3 absorption in perfused proximal
tubule in vivo corresponds to the total activity of the apical Na/H
exchanger (24). Second, it is possible that the renal tubule provides a
unique environment leading to tissue-specific regulation of NHE3
related, for example, to different PKC isoform profiles or differential
activation of isoforms in the proximal tubule and cultured cells.
Angiotensin II (ANG II), which has a well-known dose-dependent biphasic
effect on the proximal luminal Na/H exchange activity, induces a
concentration-dependent increase in inositol trisphosphate (IP3) production and cytosolic
Ca2+, which implies a
dose-dependent increase in phospholipase C activity (22). In rat
cortical tubule suspension, low-dose ANG II
(1011 M) stimulates the
luminal Na/H exchange through a PKC-dependent pathway (14). In
contrast, high-dose ANG II
(10
7 M) inhibits the
luminal Na/H exchange activity through phospholipase A2
(PLA2) and cytochrome
P-450-dependent metabolites of
arachidonate, likely 5,6-epoxyeicosatrienoic acid, and the inhibition
of the latter pathway unmasks the stimulatory effect of ANG II (14). Whether the stimulation of specific PKC isoforms by high-dose ANG II
might contribute to the stimulation of
PLA2 is not known (14).
Therefore the aim of the present study was to determine the effects of acute phorbol ester and ANG II pretreatment of the cortical tubule suspension on the Na/H activity and abundance of PKC isoforms in LMV.
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MATERIALS AND METHODS |
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Preparation of partially purified LMV.
A suspension of cortical tubules free of glomeruli was prepared as
previously described (22). Five-milliliter samples of tubule suspension
in a Ringer medium (in mM: 116 NaCl, 3 KCl, 1 MgSO4, 0.2 KH2PO4,
0.8 K2HPO4, 10 HEPES, 1 CaCl2, 25 NaHCO3, 5 glucose, 5 alanine, 10 sodium pyruvate, and 0.1% bovine serum albumin) were equilibrated at 37°C under an atmosphere of 95%
O2-5%
CO2 for 15 min before the addition
of the agent that had to be tested. The incubation was stopped by
adding 10 ml of ice-cold Ringer medium. After centrifugation, the
tubules were resuspended in hyposmotic homogenization medium [in
mM: 125 mannitol, 2 dithiothreitol, 5 Trizma, pH 7.4, 10 EGTA-Tris, pH
7.4, 10 benzamidine, 0.2 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] with 0.1 µg/ml aprotinin and 12.5 µg/ml leupeptin, 180 mosmol/kgH2O.
Purified LMV were prepared by Mg2+
precipitation as described in (4). The final pellet was resuspended at
2-4 µg/µl in homogenization medium for Western blot
experiments. For 22Na uptake
experiments, LMV were resuspended in a Na-free medium [in mM: 200 mannitol, 3 EGTA, 50 tetramethylammonium (TMA) nitrate, and 50 Tris-MES, pH 6], pelleted (30,000 g for 30 min, 4°C), resuspended in
the same acidic medium, and kept at 80°C until use. The
purity and yield of the LMV preparation were routinely followed by
measuring the activity of enzyme markers maltase (10) and
Na+-K+-ATPase
(11). There was no difference between the membranes prepared from
control tubules and those pretreated with phorbol esters or ANG II,
regarding the specific activity of the markers (not shown) as well as
yield [33 ± 3 and 32 ± 3% for maltase, and 6 ± 1 and
6 ± 1% for
Na+-K+-ATPase
of controls and phorbol myristate acetate (PMA), respectively; 44 ± 3 and 43 ± 3% for maltase, and 4.9 ± 0.5 and 5.3 ± 0.6% for Na+-K+-ATPase
of controls and ANG II, respectively] or enrichment (10 ± 1 and 9 ± 1 for maltase, and 1.8 ± 0.1 and 2.0 ± 0.1 for
Na+-K+-ATPase
of controls and PMA, respectively; and 13 ± 1 and 13 ± 1 for
maltase, and 1.5 ± 0.1 and 1.7 ± 0.2 for
Na+-K+-ATPase
of controls and ANG II, respectively).
Immunoblot analysis. Aliquots of 90 µl of LMV fractions were mixed with 30 µl of Laemmli buffer, heated
at 90°C for 10 min and stored at 20°C until use.
Aliquots were subjected to SDS-PAGE (7.5%) as described by Laemmli
(17); equivalent amounts of protein from controls or experimentals were
run in parallel. Each sample was run in duplicate. Proteins on the gel
were electrophoretically transferred onto nitrocellulose membranes
(Schleicher & Schuell, 0.45 mm) by using a Bio-Rad apparatus. The blots
were rinsed and incubated with affinity-purified anti-NHE2 or anti-NHE3
polyclonal antibodies (1/1,000 and 1/300, respectively) (9, 2) or
anti-PKC isozyme antibodies (Santa Cruz for
,
, and
; Life
Technologies for
) diluted (1/1,000 for
and
, 1/500 for
,
and 1/1,500 for
). After 1 h at room temperature for all antibodies
used except PKC-
(overnight at 4°C), the nitrocellulose
membranes were washed and probed with horseradish peroxidase-conjugated goat anti-rabbit antibody and then developed with an enhanced chemiluminescence kit (ECL) from Amersham. Polaroid pictures were taken
with an Amersham apparatus. Apparent molecular masses were calculated
on the basis of the mobility of a panel of molecular mass markers from
Sigma. Quantitative data were obtained by scanning the photos
(Hewlett-Packard Scanjet CX using Deskan) and analyzed with NIH image software.
22Na uptake. Na/H activity was assessed by proton gradient-stimulated initial rate of 22Na uptake using the rapid filtration method (pHin = 6.0, pHout = 8.0). Reaction was initiated by adding 20 µl of LMV (2-4 µg protein/µl in the Na-free acidic resuspension medium described above) to 80 µl of uptake medium (in mM: 1 22NaCl, 0.2 µCi/µl, 200 mannitol, 3 EGTA, 50 TMA nitrate, and 50 Tris-HEPES, pH 8) with or without inhibitors [ethylisopropylamiloride (EIPA 100 µM) and Hoe-694 (100 µM or 1 µM)]. It was stopped 10 s later by adding 2.5 ml of ice-cold washing solution (in mM: 280 mannitol, 20 Tris-HEPES, pH 7.4, and 0.5 amiloride), and the mixture was filtered onto nitrocellulose filter (Millipore). The filters were rinsed three times with 5 ml of washing solution and counted by liquid scintillation. Each sample was assayed in triplicate at room temperature. 22Na uptake was linear with time until 30 s (not shown).
Materials. Collagenase was obtained from Boehringer; bovine serum albumin, phorbol myristate acetate, phorbol didecanoate, ANG II, amiloride, and EIPA were from Sigma; bisindolylmaleimide (GF109203X) was obtained from Calbiochem. Hoe-694 was a gift from Dr. L. Counillon from Sofia-Antipolis University France. Statistics. Results are expressed as means ± SE. Statistical significance was assessed by Student's t-test. Paired t-tests were used to analyze 22Na uptake data. ![]() |
RESULTS |
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Identification of NHE proteins and NHE transport
activities in LMV isolated from cortical tubule
suspensions. NHE proteins present in LMV were
characterized by Western blot analysis (Fig. 1, A and
B). Under basal conditions,
anti-NHE3 antibodies, whose specificity was documented in a previous
study (2), were used to confirm the presence of NHE3 in the LMV.
Anti-NHE3 antibodies reacted with 83- to 85-kDa protein. Anti-NHE2
antibodies, whose specificity was also documented in a previous study
(9), reacted with a 85-kDa protein. The presence of NHE2 protein in the
preparation represents a contamination with distal cortical structures,
cortical thick ascending limb and distal convoluted
tubule, where NHE2 protein is present (9). No
immunoreactivity was detected with anti-NHE1 antibodies (data not
shown).
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Na/H exchange activity in LMV was assessed by the initial rate of 22Na uptake in the presence of an outward H+ gradient. In preliminary experiments with LMV, we found an IC50 value of 10 µM EIPA, and we used 100 µM EIPA to completely inhibit the Na/H exchange activity as documented by others (21). As shown in Fig. 1C, LMV total Na uptake was markedly inhibited by 100 µM EIPA (81% inhibition) and was almost completely resistant to 100 µM Hoe-694 (7% inhibition), which was previously documented to completely inhibit NHE2 activity and marginally NHE3 (29, 26). These results supported that EIPA-sensitive Na uptake represented Na/H exchange activity due to NHE3 in LMV. However, to be sure that the small Hoe-694-sensitive Na uptakewas not due to the NHE2 protein present in the preparation and located in the distal structures, we tested our ability to measure the transport activity of other transport proteins, the cotransporters BSC1 (bumetanide sensitive) and TSC (thiazide sensitive), also present in the preparation and located in cortical distal structures (9). No component of 22Na uptake inhibited by bumetanide or thiazide was observed in LMV incubated with 100 mM KCl. Actually, KCl did not elicit 22Na uptake different from that measured in LMV incubated without KCl (data not shown), whereas we were able to measure BSC1 transport activity in basolateral membranes of medullary thick ascending limb with the same protocol (23). It should be noted that no component sensitive to 1 µM Hoe-694, i.e., NHE1 activity, was present, confirming with Western blot analysis that LMV were not contaminated with basolateral membranes. Finally, NHE4 activity, which is a basolateral isoform (8), with amiloride sensitivity close to that of NHE3, could not have contributed to the Hoe-694-resistant Na uptake.
Effects of PMA on NHE3 protein abundance and Na/H
exchange activity in LMV. As shown in Fig.
1B, NHE3 protein abundance in LMV was
unchanged after PMA treatment of the suspension (108.4 ± 15.1% of
controls, n = 4). The values of Na
uptake in LMV when cortical tubules were previously exposed to PMA
(107 M for 4 min)
are shown in Table 1. Total Na uptake was
enhanced by 9.6 ± 3.8% (n = 11, P < 0.02), and the residual
EIPA-resistant component of Na uptake did not change. EIPA-sensitive Na
uptake increased by 12 ± 3.5%
(n = 11, P < 0.001). Thus PMA increased NHE3
activity in LMV.
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Preincubation of the cortical tubule suspension with specific PKC
inhibitor bisindolylmaleimide
(105 M for 5 min) followed
by an additional 4-min incubation with PMA resulted in a complete
inhibition of the PMA effect on EIPA-sensitive Na uptake (Table 1).
EIPA-sensitive Na uptake was similar in controls and
bisindolylmaleimide-pretreated tubules (not shown).
Effect of pretreatment of cortical tubules with PMA on
PKC isoform abundance in LMV. As shown in Fig.
2, proteins immunoreacting with anti-PKC
isoforms ,
,
, and
were detected in LMV isolated from
control tubules, and the abundance of PKC isoforms
,
, and
was increased in LMV isolated from tubules incubated with PMA (140 ± 11, 182 ± 24, and 133 ± 8% of controls for
,
and
, respectively, n = 10). The
inactive phorbol didecanoate had no effect (Fig. 2).
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Effects of pretreatment of cortical suspensions
with low-dose ANG II (1011 M)
on Na/H exchange activity and PKC isoform abundance in LMV.
The values of Na uptake in LMV when cortical tubule suspensions were
previously incubated with
10
11 M ANG II for 4 min are
shown in Table 2. Total Na uptake increased by 10.0 ± 2.3% (n = 7, P < 0.001), and the residual
EIPA-resistant component of Na uptake did not change. EIPA-sensitive Na
uptake increased by 11 ± 2.6% (n = 7, P < 0.001). Thus low-dose ANG
II increased NHE3 activity. Preincubation of cortical tubules with bisindolylmaleimide resulted in a complete inhibition of the ANG II
effect on EIPA-sensitive Na uptake (Table 2).
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Effect of pretreatment of cortical tubules with
high-dose ANG II on Na/H exchange activity in LMV.
As shown in Fig. 4, incubation of the
cortical tubule suspension with
107 M ANG II (10 min)
decreased the EIPA-sensitive Na uptake by 13.4 ± 3.4% (P < 0.02, n = 5). This inhibitory
effect of ANG II was observed when the tubules were preincubated with
bisindolylmaleimide (10
5 M,
5 min). Thus 10
7 M ANG II
inhibited NHE3 activity in LMV, but this effect was not PKC mediated.
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DISCUSSION |
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The present study was designed to identify the Na/H antiporter isoforms
present in the luminal membranes isolated from rat kidney cortical
tubules and to examine the effects of acute pretreatment with PMA or
ANG II on NHE activity and PKC isoform abundance. The main informative
points are the following.
1) In LMV, the Na/H exchange
activity observed was attributed to NHE3.
2) PMA pretreatment increased LMV
Na/H exchange activity while NHE3 protein abundance remained unchanged
in LMV. PMA increased abundance of PKCs isoforms ,
, and
in
LMV. PKC inhibition suppressed the PMA effects.
3) Low-dose ANG II increased LMV
Na/H exchange activity and only PKC-
abundance in LMV. PKC
inhibition suppressed the low-dose ANG II effects. High-dose ANG II
decreased LMV Na/H exchange activity. PKC inhibition did not affect the
high-dose ANG II effects.
The presence of NHE3 protein and activity in LMV was expected, since NHE3 is well-known as the main luminal isoform in proximal tubule cells (5). The present study also shows the presence of NHE2 protein, in agreement with previous observations reporting the presence of NHE2 protein in LMV isolated from rat kidney cortex (9, 13). However, Na/H exchange activity in LMV represented NHE3 activity. Indeed, the very small Hoe-694-sensitive transport activity reflects the marginal inhibition of NHE3 activity in agreement with recent data in a similar preparation (29). Indeed, NHE2 protein, mRNA, and transport activity have not been found in the proximal tubule (9, 12, 26). The contamination of the preparation by distal structures is not sufficient to give measurable transport activity, in agreement with previous findings that ~97% of the total luminal surface of tubules in rat renal cortex is brush-border membrane surface (16). The presence in LMV of the two other NHE isoforms described in the kidney, NHE1 and NHE4, may be ruled out, since the latter isoforms are present on the basolateral membranes (6, 8). In addition, the absence of 1 µM Hoe-694-sensitive Na uptake and NHE1 protein in LMV confirmed the absence of contamination of LMV by basolateral membrane vesicles.
PMA acutely increased NHE3 activity, and protein abundance remained
unchanged in LMV. The effects of PMA on NHE3 were the net result of
increased abundance and activity of PKC isoforms ,
, and
. The
present study showing an increase of the luminal Na/H exchange activity
by PMA is in agreement with the previous results obtained by acutely
applying phorbol esters basolaterally or luminally in rat proximal
tubule perfused in vivo (19, 25) or by directly applying phorbol esters
on LMV isolated from rabbit kidney cortex (27). PMA-induced PKC
activation could directly increase the activity of the transporter,
since Weinman and his group (28) have shown that PKC-mediated
phosphorylation of solubilized luminal membrane proteins elicited a
rise in Na/H activity subsequently measured in reconstituted liposomes
containing the phosphorylated proteins. However, our results are not in
agreement with previous studies using phorbol ester on proximal
tubular-like cell lines (20) or in NHE-deficient fibroblast line
transfected with NHE3 (15, 18). In the latter studies, phorbol ester
acutely decreased NHE3 activity. A possible explanation for this
discrepancy may be that the regulation of NHE3 by phorbol esters could
be tissue specific, related to different PKC isoform profiles or
PKC-specific binding or anchoring proteins in proximal tubules and
cultured cells. The loss of a putative cytosolic regulator during the
preparation of LMV cannot be excluded but seems less probable with
regard to the stimulation of luminal Na/H exchange activity by phorbol esters observed in intact tubules (19, 25).
Low-dose ANG II (1011 M)
increased the Na/H exchange activity in LMV, and this effect was
suppressed by PKC inhibition, in agreement with our previous studies in
cortical tubule suspensions (14). In contrast to the PMA data, ANG II
selectively increased PKC-
abundance in LMV, whereas the abundance
of PKC isoforms
,
, and
remained unchanged. The absence of
modification of the latter PKC isoforms by low-dose ANG II, i.e., the
calcium and diacylglycerol (DAG)-sensitive
-isoform
and the DAG-sensitive
- and
-isoforms, is compatible with the
lack of stimulation of phospholipase C as indicated by the unchanged
IP3 production and cytosolic
Ca2+ observed in our previous
studies with cortical tubule suspensions (22). We have no indication in
the present study on the mechanisms of stimulation of the calcium- and
DAG-insensitive PKC-
by low-dose ANG II. Taken together, the
low-dose ANG II and PMA data suggest that NHE3 activity may be
stimulated by increase in PKC-
alone and by a simultaneous increase
in PKC isoforms
,
, and
.
In conclusion, PMA acutely increases the NHE3 activity, by
translocation of PKC isoforms ,
, and
into LMV, whereas
low-dose ANG II stimulated NHE3 activity via selective translocation of PKC-
. High-dose ANG II inhibited Na/H exchange activity in LMV, and
this was not PKC mediated.
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ACKNOWLEDGEMENTS |
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Portions of this work were presented at the 28th, 29th, and 30th Meetings of the American Society of Nephrology and have been published in abstract form (J. Am. Soc. Nephrol. 6: 743, 1995).
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FOOTNOTES |
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R. Chambrey was supported by Fondation pour la Recherche Médicale. This work was partially supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-19407.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Poggioli, INSERM U356, 15 rue de l'École de Médecine 75270 Paris Cédex 06 France (E-mail: poggioli{at}ccr.jussieu.fr).
Received 25 November 1998; accepted in final form 30 June 1999.
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