1 Department of Physiology, University of Zurich-Irchel, CH-8057 Zurich; 4 Renal Division, University Hospital, CH-8091 Zurich, Switzerland; and 2 Center of Mineral Metabolism and Clinical Research and 3 Division of Nephrology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Insulin is an important regulator of renal salt and water excretion, and hyperinsulinemia has been implicated to play a role in hypertension. One of the target proteins of insulin action in the kidney is Na+/H+ exchanger 3 (NHE3), a principal Na+ transporter responsible for salt absorption in the mammalian proximal tubule. The molecular mechanisms involved in activation of NHE3 by insulin have not been studied so far. In opossum kidney (OK) cells, insulin increased Na+/H+ exchange activity in a time- and concentration-dependent manner. This effect is due to activation of NHE3 as it persisted after pharmacological inhibition of NHE1 and NHE2. In the early phase of stimulation (2-12 h), NHE3 activity was increased without changes in NHE3 protein and mRNA. At 24 h, enhanced NHE3 activity was accompanied by an increase in total and cell surface NHE3 protein and NHE3 mRNA abundance. All the effects of insulin on NHE3 activity, protein, and mRNA were amplified in the presence of hydrocortisone. These results suggest that insulin stimulates renal tubular NHE3 activity via a biphasic mechanism involving posttranslational factors and an increase in NHE3 gene expression and the effects are dependent on the permissive action of hydrocortisone.
kidney; sodium transport; hormones
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DIABETES MELLITUS IS ASSOCIATED with Na+ and water retention and extracellular fluid volume expansion (9, 48). A principal site of renal salt and water reabsorption is the proximal tubule, where insulin receptors have been found in different species (16, 46, 61). Insulin is present in the plasma and glomerular ultrafiltrate and is degraded in the proximal tubule (32). Several studies have provided evidence that insulin decreases urinary Na+ excretion (24, 45, 47, 54, 57). Baum (10) has shown that insulin directly stimulates volume absorption in rabbit proximal convoluted tubules. The stimulatory effect on the proximal tubule is associated with increased apical H+ secretion (40, 60) and EIPA-sensitive Na+ uptake (28), findings compatible with increased apical membrane Na+/H+ exchange activity. One postulate is that peripheral insulin resistance may be associated with relatively preserved insulin sensitivity in the kidney, and the price of hyperinsulinemia are renal NaCl retention and salt-sensitive hypertension (51, 56).
In the mammalian proximal tubule, >60% of the Na+
absorption is mediated by apical brush-border membrane
Na+/H+ exchange. Of the seven isoforms known to
date, NHE3 is the only Na+/H+ exchanger isoform
definitively shown to be expressed in the brush-border membrane of the
renal proximal tubule, on the basis of antigenic (6, 13)
and functional data (20, 62, 67). NHE3 mediates proximal
tubule transcellular NaCl absorption via coupled transport with
Cl/base exchange (8, 63) as well as
paracellular NaCl transport by lowering luminal HCO
concentration
(52). The importance of NHE3 in sustaining extracellular fluid volume is evident by the hypovolemia and hypotension seen in NHE3
null mice (55). Previous studies examining the effect of
insulin on the proximal tubule did not specifically address the NHE3
isoform. The present study investigates the effects of insulin on
apical membrane NHE3 activity, surface protein, total protein, and
transcript levels in a cell line of the opossum kidney with proximal
tubule characteristics (OKP cells). Because hydrocortisone has been
shown to exert a permissive effect on the acid-induced activation of
Na+/H+ exchange activity (5), we
also examined glucocorticoid dependence of insulin-induced activation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials and supplies. All chemicals were obtained from Sigma (St. Louis, MO), except for the following: acetoxymethyl derivative of 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes, Eugene, OR); NHS-SS-biotin and immobilized streptavidin (Pierce, Rockford, IL); and culture media (GIBCO BRL, Grand Island, NY).
Cell culture.
OKP cells (22) were passaged in high-glucose (450 mg/dl)
DMEM supplemented with 10% FBS, penicillin (100 U/ml), and
streptomycin (100 µg/ml). Before study, confluent cells were rendered
quiescent by incubation in serum-free media [1:1 mixture of
low-glucose (100 mg/dl) DMEM and Ham's F-12 ± 106
M hydrocortisone] for 24-48 h. Human insulin (10
6
to 10
10 M) was applied for the stated period of time
before the assays.
Measurement of intracellular pH and
Na+/H+
exchange activity.
Continuous measurement of cytoplasmic pH (pHi) was
accomplished using the intracellularly trapped pH-sensitive dye BCECF, as described previously (4). Cells were loaded with 10 µM acetoxymethyl ester of BCECF for 35 min at 37°C, and
pHi was estimated from the ratio of fluorescence
(ex: 500 and 450 nm,
em: 530 nm, where ex is excitation and em is emission) in a computer-controlled spectrofluorometer (8000C, SLM Instruments, Urbana, IL, and RF-5000, Shimadzu, Kyoto, Japan). The BCECF excitation fluorescence
ratio was calibrated intracellularly using K/nigericin as described (3). Na+/H+ exchange activity was
assayed as the initial rate of the Na+-dependent
pHi increase after an acid load in the absence of
CO2/HCO3, and results are reported as
dpHi/dt. Comparisons were always made between
cells of the same passage studied on the same day. Intracellular buffer
capacity was measured by pulsing with 20 mM NH4Cl. Buffer capacity (
) was then calculated according to the formula
= [NH4Cl]/
pHi. Results for control and
insulin-treated cells were not significantly different (34.5 vs. 34.4 mM, respectively).
NHE3 antigen. Cells were rinsed three times with ice-cold PBS and Dounce-homogenized in isotonic Tris-buffered saline (150 mM NaCl, 50 mM Tris · HCl, pH 7.5, 5 mM EDTA) containing proteinase inhibitors [100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 4 µg/ml aprotinin, 4 µg/ml leupeptin]. After nuclei removal (13,000 g, 4°C , 5 min; Eppendorf 5415C, Hamburg, Germany), membranes were pelleted (109,000 g, 4°C, 20 min; Sorvall RCM 120EX, rotor S120 AT2-0130, Sorvall Products, DuPont, Wilmington, DE) and resuspended in Tris-buffered saline, and total protein content was determined by the method of Bradford. Fifteen micrograms of protein were diluted 1:5 in 5× SDS loading buffer (1 mM Tris · HCl, pH 6.8, 1% SDS, 10% glycerol, 1% 2-mercaptoethanol), size fractionated by SDS-PAGE (7.5% gel), and electrophoretically transferred to nitrocellulose. After blocking for 1 h (5% nonfat milk, 0.05% Tween 20 in PBS), blots were probed in the same buffer with a polyclonal anti-opossum NHE3 antibody [antiserum 5683, generated against a maltose-binding protein/NHE3 (amino acids 484-839) fusion protein] at a dilution of 1:300 (4). Blots were washed in 0.05% Tween 20 in PBS once for 15 min and twice for 5 min, incubated with a 1:10,000 dilution of peroxidase-labeled sheep anti-rabbit IgG, washed as above, and then visualized by enhanced chemiluminescence. NHE3 protein abundance was quantitated by densitometry (BioCapt software version 72.02s for Windows, Vilbert Lourmat, Marne la Vallée, France) and Scion Image Beta 3b, 1998 (Scion, Frederick, MD).
To measure plasma membrane NHE3, we used a surface biotinylation assay (23). Monolayers were rinsed three times with ice-cold PBS-Ca-Mg (PBS with 0.1 mM CaCl2, 1.0 mM MgCl2). Membrane proteins were then biotinylated by incubation of cells in 1.5 mg/ml NHS-SS-biotin in 10 mM triethanolamine (pH 7.4), 2 mM CaCl2, and 150 mM NaCl for 90 min at 4°C. After labeling, plates were washed with 6 ml quenching buffer (PBS-Ca-Mg, with 100 mM glycine) for 20 min at 4°C. Cells were then lysed in RIPA buffer (150 mM NaCl, 50 mM Tris · HCl, pH 7.4, 5.0 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin), extracts were rocked for 30 min at 4°C and centrifuged at 12,000 g at 2°C for 10 min, and the supernatant was diluted to 3 mg/ml with RIPA buffer. Biotinylated proteins were then affinity precipitated with streptavidin-conjugated agarose, released byNHE3 transcript.
RNA was extracted using RNeasy (Qiagen, Valencia, CA). Fifteen
micrograms of total RNA were size fractionated by agarose-formaldehyde gel electrophoresis and transferred to nylon membranes. The
radiolabeled NHE3 probe was synthesized from full-length OKP NHE3 cDNA
(7) and an 18S probe from a 752-base
SphI/BamHI fragment of mouse 18S rRNA (no. 63178, American Type Culture Collection, Rockville, MD) by the random hexamer
method. Prehybridization, hybridization, and washing were performed as
described previously (4). Filters were exposed to film
overnight at 70°C, and labeling was quantitated by densitometry.
Changes in NHE3 abundance were normalized for changes in 18S rRNA abundance.
Statistics. All results are reported as means ± SE. Statistical analysis was performed using ANOVA unless stated otherwise, and n refers to the number of plates studied.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Insulin increases
Na+/H+
exchanger activity in OKP cells.
A typical tracing (Fig. 1A)
shows that insulin stimulates
Na+/H+ exchange activity. Figure 1B
shows the time course of the insulin effect. Acute incubation for 40 min does not significantly affect activity (+6%, not significant). At
2-24 h of incubation, insulin increases
Na+/H+ exchange activity. This effect was dose
dependent (Fig. 1C), with a detectable effect down to
108 M insulin and half-maximal stimulation at
~10
7 M for both the acute (2 h) and chronic (24 h)
effect. OKP cells express an EIPA-resistant
Na+/H+ exchanger that is encoded by NHE3
(7). However, to exclude the possibility that the observed
changes in dpHi/dt may be mediated through an
effect of insulin on another NHE isoform, we performed experiments in
the presence of 100 µM HOE-642, which completely inhibits NHE1 or
NHE2, but not NHE3. HOE-642 does not affect baseline or
insulin-stimulated Na+/H+ exchange activity
(Fig. 2), suggesting that the observed
effect of insulin on Na+/H+ exchange is
exclusively on NHE3.
|
|
|
Insulin increases total and cell surface NHE3 protein abundance.
Changes in NHE3 activity can be associated with changes in total
cellular NHE3 protein and/or changes in surface plasma membrane NHE3
protein. Figure 4A shows a
typical blot depicting the effect of insulin in the presence or absence
of 106 M hydrocortisone on OKP NHE3 total and cell
surface protein abundance. Insulin does not affect total or cell
surface NHE3 protein abundance after 12 h when NHE activity is
clearly stimulated. In contrast, insulin at 24 h increased total
cellular NHE3 antigen by 27% and surface NHE3 by 60%. The results are
summarized in Fig. 4B. These results indicate that the early
(8-12 h) and late (24 h) stimulation of NHE is mediated by
distinct mechanisms. In the absence of hydrocortisone, the increase in
cellular and surface NHE3 is variable and much less pronounced (Fig.
4A). In the presence of hydrocortisone, the increase in NHE3
activity observed at 24 h is associated with increased total cell
and surface antigen.
|
Insulin increases NHE3 transcript.
Insulin treatment of OKP cells for 24 h increases NHE3 transcript
abundance (Fig. 5A). In
contrast, insulin treatment for 12 h actually slightly decreases
the level of NHE3 transcript (P = 0.021). Again, we
determined the hydrocortisone dependence of the insulin effect on NHE3
transcript level at 24 h. Insulin alone increases NHE3 transcript
level slightly by ~43%. As shown before (5),
hydrocortisone (106 M) by itself approximately doubles
the NHE3 transcript level. Combined treatment with insulin and
hydrocortisone results in another 2.4-fold increase in NHE3 mRNA
compared with hydrocortisone alone.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The role of insulin in renal tubular salt and water handling has been previously implicated from clinical observations (24, 47, 54) and tubule perfusion studies in animals (10, 40, 60). The causal relationship between hyperinsulinemia and hypertension is still an issue of debate (30). Reaven (51) has suggested that even in states of hyperinsulinemia, additional factors other than insulin likely contribute to hypertension. Although it is controversial as to whether hyperinsulinemia leads to salt-sensitive hypertension, the present body of data are strongly supportive of a salt-retaining action of insulin on the kidney. Insulin stimulates Na+ transporters and Na+ absorption at both the proximal tubule (11, 28, 40, 60) and thick ascending limb (39, 60).
The molecular mechanisms of the insulin-induced increase in
Na+ transport have not been examined. The present study
demonstrates that insulin directly stimulates the
Na+/H+ exchanger NHE3 in OKP cells in a time-
and concentration-dependent manner. The concentrations used in our
experiments were higher than the circulating plasma levels. Therefore,
we cannot exclude that some of the effects on the
Na+/H+ exchanger NHE3 are mediated through the
insulin-like growth factor-1 receptor. However, for NHE3 activity,
significant stimulation by insulin was detectable down to a
concentration of 108 M. The NHE1 isoform is ubiquitously
expressed in the kidney (14), and there is indirect
evidence supporting stimulation of NHE1 by insulin in cultured renal
cells (27). Although NHE2 is expressed in the kidney
(19, 59), its functional role is still enigmatic (20) and may only be activated under certain
circumstances. We have ruled out the role of both NHE1 and NHE2 in
mediating the insulin-induced increase in
Na+/H+ exchange activity. We showed that
insulin specifically upregulates proximal tubule NHE3, which likely
mediates the increased proximal tubule Na+ absorption in
response to insulin. The stimulation of NHE3 by insulin has two
characteristics. First, it occurs in a biphasic fashion. Second, it is
amplified by glucocorticoids.
Na+/H+ exchangers are regulated by a wide variety of agonists through vastly different mechanisms. Regulation at the level of transcription (4, 5, 7, 11, 17, 18, 37), translation (67), protein trafficking (2, 21, 23, 25, 26, 33, 35, 36, 41, 43, 49, 68-71), phosphorylation (42, 50, 64-66, 72, 73), and binding to protein (12) or lipid regulators (1) has been implicated or proven. A single condition or agonist can regulate NHE3 at more than one step. This has been shown for acid incubation (4, 5, 7, 67, 68), parathyroid hormone (23, 26), and dopamine (33, 65). The induction of NHE3 activation by insulin is time dependent, as a significant increase in dpHi/dt was detectable only at 2 h and beyond. After 12 h, NHE3 activity is clearly increased whereas surface NHE3 protein abundance is still unchanged in insulin-treated cells. The possibility remains that the dpHi/dt assay is more sensitive than the biotinylation assay. Alternatively, a more plausible explanation is that other posttranslational mechanisms may be operative and contribute to the stimulation of Na+/H+ exchange by insulin (23, 26, 44). A biphasic response has previously been described for parathyroid hormone (23, 26) and dopamine (33, 65) involving changes in transport activity of surface NHE3 followed by internalization of NHE3 protein. However, in those two situations, the decrease in NHE3 surface protein commences after a relatively short time. In the case of insulin, surface NHE3 activity is increased without changes in surface NHE3 protein for over 12 h. At present, the mechanism of how insulin induces and sustains this suppression of surface NHE3 transporters is unknown. After 24 h of incubation with insulin, one can see concomitant increases in surface and total NHE3 protein abundance that approximate but are not equal to the magnitude of increase in NHE3 activity. The slightly higher increase in surface NHE3 compared with total NHE3 may reflect an additional step, whereas the increased cellular pool of NHE3 is preferentially targeted to the cell membrane. Moreover, an increase in NHE3 protein is associated with an increase in NHE3 mRNA at 24 h. This pattern of coordinated upregulation at the levels of activity, surface protein, total protein, and mRNA is reminiscent of the effects of thyroid hormone on NHE3 (18).
The stimulation of intrinsic NHE3 activity in the early phase and the
increase in NHE3 activity, protein, and mRNA in the late phase are all
enhanced by hydrocortisone. At 109 M, where
glucocorticoid itself has no effect on NHE3 activity (11),
the presence of glucocorticoid allows insulin to exert its full action
on NHE3, hence befitting the classic permissive role described in the
pioneering manuscript by Ingle (34) a half-century ago. At
10
7 and 10
8 M, when corticosteroids
themselves activate NHE3, the presence of insulin further increases
NHE3 activity. At this point, hydrocortisone acts more like a
biological amplifier as discerned by Granner (29). In a
saturating dose of hydrocortisone (10
5 M), the addition
of insulin no longer leads to further stimulation. Whether this is
synergism, permission, or amplification, the interactive relationship
(both positive and negative) between glucocorticoids and a variety of
other agonists is pervasive in mammalian biology (53). In
the liver, the ability of glucocorticoids to promote hepatic glycogen
synthesis is "proinsulin" (58). In contrast, in
skeletal muscle, glucocorticoid decreases the ability of insulin to
stimulate glycogen synthesis (15). In the kidney, the
acid-induced increase in Na+/H+ exchange can be
abolished by adrenalectomy (38). We have shown that this
is a direct effect of glucocorticoids because the acid-induced increase
in NHE3 is dependent on the presence of hydrocortisone in the cell
culture media during serum deprivation and acid incubation (5,
31). Glucocorticoids may represent a more generally permissive agent for regulation of NHE3 in the kidney. The mechanism of the permissive effect of glucocorticoids is presently unknown.
In summary, we have shown that insulin activates the Na+/H+ exchanger NHE3 in OKP cells. This effect is biphasic in nature, with distinct mechanisms that involve increased activity of existing NHE3 proteins on the cell surface followed later by increased NHE3 transcript and total cellular and surface NHE3 protein. In both phases, the insulin-stimulated increase in NHE3 is enhanced by the presence of glucocorticoids. In conjunction with data from clinical and tubule perfusion studies, we propose that insulin stimulates NHE3 and proximal tubule Na+ absorption and contributes to the volume expansion and hypertension seen in insulin-resistance states.
![]() |
ACKNOWLEDGEMENTS |
---|
P. M. Ambühl was supported by a grant from the Swiss National Science Foundation (31-54957.98) and the Hermann Klaus Foundation. O. W. Moe was supported by the American Heart Association Texas Affiliate (98G-052), National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-48482, R01-DK-54396, and PO1-DK-20543, the Department of Veterans Affairs Research Service, and a Seed Grant from the Center of Mineral Metabolism and Clinical Research. D. Fuster was supported by the Swiss National Science Foundation.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: P. M. Ambühl, Renal Division, Univ. Hospital, Rämistrasse 100, CH-8091 Zürich, Switzerland (E-mail: patrice.ambuehl{at}dim.usz.ch).
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.
March 26, 2002;10.1152/ajprenal.00365.2001
Received 13 December 2001; accepted in final form 25 March 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aharonovitz, O,
Zaun HC,
Balla T,
York JD,
Orlowski J,
and
Grinstein S.
Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate.
J Cell Biol
150:
213-224,
2000
2.
Akhter, S,
Cavet ME,
Tse CM,
and
Donowitz M.
C-terminal domains of Na(+)/H(+) exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger.
Biochemistry
39:
1990-2000,
1990.
3.
Alpern, RJ.
Mechanism of basolateral membrane H+/OH/HCO
4.
Ambühl, P,
Amemiya M,
Preisig PA,
Moe OW,
and
Alpern RJ.
Chronic hyperosmolality increases NHE3 activity in OKP cells.
J Clin Invest
101:
170-177,
1998
5.
Ambühl, PM,
Yang X,
Peng Y,
Preisig PA,
Moe OW,
and
Alpern RJ.
Glucocorticoids enhance acid activation of the Na+/H+ exchanger 3 (NHE3).
J Clin Invest
103:
429-435,
1999
6.
Amemiya, M,
Loffing J,
Lotscher M,
Kaissling B,
Alpern RJ,
and
Moe OW.
Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb.
Kidney Int
48:
1206-1215,
1995[ISI][Medline].
7.
Amemiya, M,
Yamaji Y,
Cano A,
Moe OW,
and
Alpern RJ.
Acid incubation increases NHE-3 mRNA abundance in OKP cells.
Am J Physiol Cell Physiol
269:
C126-C133,
1995
8.
Aronson, PS.
Ion exchangers mediating NaCl transport in the proximal tubule.
Wien Klin Wochenschr
109:
435-440,
1997[ISI][Medline].
9.
Bank, N,
and
Aynedjian HS.
Progressive increases in luminal glucose stimulate proximal sodium absorption in normal and diabetic rats.
J Clin Invest
86:
309-316,
1990[ISI][Medline].
10.
Baum, M.
Insulin stimulates volume absorption in the rabbit proximal convoluted tubule.
J Clin Invest
79:
1104-1109,
1987[ISI][Medline].
11.
Baum, M,
Amemiya M,
Dwarakanath V,
Alpern RJ,
and
Moe OW.
Glucocorticoids regulate NHE-3 transcription in OKP cells.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F164-F169,
1996
12.
Biemesderfer, D,
DeGray B,
and
Aronson PS.
Active (9.6s) and inactive (21s) oligomers of NHE3 in microdomains of the renal brush border.
J Biol Chem
276:
10161-10167,
2001
13.
Biemesderfer, D,
Pizzonia J,
Abu-Alfa A,
Exner M,
Reilly R,
Igarashi P,
and
Aronson PS.
NHE3: a Na+/H+ exchanger isoform of renal brush border.
Am J Physiol Renal Fluid Electrolyte Physiol
265:
F736-F742,
1993
14.
Biemesderfer, D,
Reilly RF,
Exner M,
Igarashi P,
and
Aronson PS.
Immunocytochemical characterization of Na+-H+ exchanger isoform NHE-1 in rabbit kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F833-F840,
1992
15.
Bjorntorp, P.
Neuroendocrine perturbations as a cause of insulin resistance.
Diabetes Metab Res Rev
15:
427-441,
1999[ISI][Medline].
16.
Blanchard, RF,
Davis PJ,
and
Blas SD.
Physical characteristics of insulin receptors on renal cell membranes.
Diabetes
27:
88-95,
1978[ISI][Medline].
17.
Cano, A.
Characterization of the rat NHE3 promoter.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F629-F636,
1996
18.
Cano, A,
Baum M,
and
Moe OW.
Thyroid hormone stimulates the renal Na/H exchanger NHE3 by transcriptional activation.
Am J Physiol Cell Physiol
276:
C102-C108,
1999
19.
Chambrey, R,
Warnock DG,
Podevin RA,
Bruneval P,
Mandet C,
Belair MF,
Bariety J,
and
Paillard M.
Immunolocalization of the Na+/H+ exchanger isoform NHE2 in rat kidney.
Am J Physiol Renal Physiol
275:
F379-F386,
1998
20.
Choi, JY,
Shah M,
Lee MG,
Schultheis PJ,
Shull GE,
Muallem S,
and
Baum M.
Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule.
J Clin Invest
105:
1141-1146,
2000
21.
Chow, CW,
Khurana S,
Woodside M,
Grinstein S,
and
Orlowski J.
The epithelial Na(+)/H(+) exchanger, NHE3, is internalized through a clathrin-mediated pathway.
J Biol Chem
274:
37551-37558,
1999
22.
Cole, JA,
Forte LR,
Krause WJ,
and
Thorne PK.
Clonal sublines that are morphologically and functionally distinct from parental OK cells.
Am J Physiol Renal Fluid Electrolyte Physiol
256:
F672-F679,
1989
23.
Collazo, R,
Fan L,
Hu MC,
Zhao H,
Wiederkehr MR,
and
Moe OW.
Acute regulation of Na+/H+ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis.
J Biol Chem
275:
31601-31608,
2000
24.
DeFronzo, RA,
Cooke CR,
Andres R,
Faloona GR,
and
Davis PJ.
The effect of insulin on renal handling of sodium, potassium, calcium, and phosphate in man.
J Clin Invest
55:
845-855,
1975[ISI][Medline].
25.
D'Souza, S,
Garcia-Cabado A,
Yu F,
Teter K,
Lukacs G,
Skorecki K,
Moore HP,
Orlowski J,
and
Grinstein S.
The epithelial sodium-hydrogen antiporter Na+/H+ exchanger 3 accumulates and is functional in recycling endosomes.
J Biol Chem
273:
2035-2043,
1998
26.
Fan, L,
Wiederkehr MR,
Collazo R,
Wang H,
Crowder LA,
and
Moe OW.
Dual mechanisms of regulation of Na/H exchanger NHE-3 by parathyroid hormone in rat kidney.
J Biol Chem
274:
11289-11295,
1999
27.
Fine, LG,
Badie-Dezfooly B,
Lowe AG,
Hamzeh A,
Wells J,
and
Salehmoghaddam S.
Stimulation of Na+/H+ antiport is an early event in hypertrophy of renal proximal tubular cells.
Proc Natl Acad Sci USA
82:
1736-1740,
1985[Abstract].
28.
Gesek, FA,
and
Schoolwerth AC.
Insulin increases Na+-H+ exchange activity in proximal tubules from normotensive and hypertensive rats.
Am J Physiol Renal Fluid Electrolyte Physiol
260:
F695-F703,
1991
29.
Granner, DK.
The role of glucocorticoid hormones as biological amplifiers.
Monogr Endocrinol
12:
593-611,
1979[Medline].
30.
Hall, JE,
Brands MW,
Zappe DH,
and
Alonso Galicia M.
Insulin resistance, hyperinsulinemia, and hypertension: causes, consequences, or merely correlations?
Proc Soc Exp Biol Med
208:
317-329,
1995[Abstract].
31.
Hamm, LL,
Ambühl PM,
and
Alpern RJ.
Role of glucocorticoids in acidosis.
Am J Kidney Dis
34:
960-965,
1999[ISI][Medline].
32.
Hammerman, MR.
Interaction of insulin with the renal proximal tubular cell.
Am J Physiol Renal Fluid Electrolyte Physiol
249:
F1-F11,
1985
33.
Hu, MC,
Fan L,
Crowder LA,
Karim-Jimenez Z,
Murer H,
and
Moe OW.
Dopamine acutely stimulates Na+/H+ exchanger (NHE3) endocytosis via clathrin-coated vesicles: dependence on protein kinase A-mediated NHE3 phosphorylation.
J Biol Chem
276:
26906-26915,
2001
34.
Ingle DJ. The role of the adrenal cortex in homeostasis. J
Endocrinol : 23-37, 1952.
35.
Janecki, AJ,
Janecki M,
Akhter S,
and
Donowitz M.
Basic fibroblast growth factor stimulates surface expression and activity of Na+/H+ exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase.
J Biol Chem
275:
8133-8142,
2000
36.
Janecki, AJ,
Montrose MH,
Zimniak P,
Zweibaum A,
Tse CM,
Khurana S,
and
Donowitz M.
Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger.
J Biol Chem
273:
8790-8798,
1998
37.
Kandasamy, RA,
and
Orlowski J.
Genomic organization and glucocorticoid transcriptional activation of the rat Na+/H+ exchanger Nhe3 gene.
J Biol Chem
271:
10551-10559,
1996
38.
Kinsella, J,
Cujdik T,
and
Sacktor B.
Na+-H+ exchange activity in renal brush border membrane vesicles in response to metabolic acidosis: the role of glucocorticoids.
Proc Natl Acad Sci USA
81:
630-634,
1984[Abstract].
39.
Kirchner, KA.
Insulin increases loop segment chloride reabsorption in the euglycemic rat.
Am J Physiol Renal Fluid Electrolyte Physiol
255:
F1206-F1213,
1988
40.
Kubota, T,
Hagiwara N,
Inoue A,
and
Fujimoto M.
The effect of ouabain, insulin, and cyclic AMP on the acidification of luminal fluid in the proximal tubule of bullfrog kidney.
Jpn J Physiol
38:
549-556,
1988[ISI][Medline].
41.
Kurashima, K,
Szabo EZ,
Lukacs G,
Orlowski J,
and
Grinstein S.
Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway.
J Biol Chem
273:
20828-20836,
1998
42.
Kurashima, K,
Yu FH,
Cabado AG,
Szabo EZ,
Grinstein S,
and
Orlowski J.
Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. Phosphorylation-dependent and -independent mechanisms.
J Biol Chem
272:
28672-28679,
1997
43.
Magyar, CE,
Zhang Y,
Holstein-Rathlou NH,
and
McDonough AA.
Proximal tubule Na transporter responses are the same during acute and chronic hypertension.
Am J Physiol Renal Physiol
279:
F358-F369,
2000
44.
Moe, OW.
Acute regulation of proximal tubule apical membrane Na/H exchanger NHE-3: role of phosphorylation, protein trafficking, and regulatory factors.
J Am Soc Nephrol
10:
2412-2425,
1999
45.
Muscelli, E,
Natali A,
Bianchi S,
Bigazzi R,
Galvan AQ,
Sironi AM,
Frascerra S,
Ciociaro D,
and
Ferrannini E.
Effect of insulin on renal sodium and uric acid handling in essential hypertension.
Am J Hypertens
9:
746-752,
1996[ISI][Medline].
46.
Nakamura, R,
Emmanouel DS,
and
Katz AI.
Insulin binding sites in various segments of the rabbit nephron.
J Clin Invest
72:
388-392,
1983[ISI][Medline].
47.
Nizet, A,
Lefebvre P,
and
Crabbe J.
Control by insulin of sodium potassium and water excretion by the isolated dog kidney.
Pflügers Arch
323:
11-20,
1971[ISI][Medline].
48.
O'Hagan, M,
Howey J,
and
Greene SA.
Increased proximal tubular reabsorption of sodium in childhood diabetes mellitus.
Diabet Med
8:
44-48,
1991[ISI][Medline].
49.
Peng, Y,
Amemiya M,
Yang X,
Fan L,
Moe OW,
Yin H,
Preisig PA,
Yanagisawa M,
and
Alpern RJ.
ETB receptor activation causes exocytic insertion of NHE3 in OKP cells.
Am J Physiol Renal Physiol
280:
F34-F42,
2001
50.
Peng, Y,
Moe OW,
Chu T,
Preisig PA,
Yanagisawa M,
and
Alpern RJ.
ETB receptor activation leads to activation and phosphorylation of NHE3.
Am J Physiol Cell Physiol
276:
C938-C945,
1999
51.
Reaven, GM.
The kidney: an unwilling accomplice in syndrome X.
Am J Kidney Dis
30:
928-931,
1997[ISI][Medline].
52.
Rector, FC, Jr.
Sodium, bicarbonate, and chloride absorption by the proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
244:
F461-F471,
1983
53.
Sapolsky, RM,
Romero LM,
and
Munck AU.
How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions.
Endocr Rev
21:
55-89,
2000
54.
Saudek, CD,
Boulter PR,
Knopp RH,
and
Arky RA.
Sodium retention accompanying insulin treatment of diabetes mellitus.
Diabetes
23:
240-246,
1974[ISI][Medline].
55.
Schultheis, PJ,
Clarke LL,
Meneton P,
Miller ML,
Soleimani M,
Gawenis LR,
Riddle TM,
Duffy JJ,
Doetschman T,
Wang T,
Giebisch G,
Aronson PS,
Lorenz JN,
and
Shull GE.
Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.
Nat Genet
19:
282-285,
1998[ISI][Medline].
56.
Secchi, LA.
Mechanisms of insulin resistance in rat models of hypertension and their relationships with salt sensitivity.
J Hypertens
17:
1229-1237,
1999[ISI][Medline].
57.
Skott, P,
Vaag A,
Bruun NE,
Hother-Nielsen O,
Gall MA,
Beck-Nielsen H,
and
Parving HH.
Effect of insulin on renal sodium handling in hyperinsulinaemic type 2 (non-insulin-dependent) diabetic patients with peripheral insulin resistance.
Diabetologia
34:
275-281,
1991[ISI][Medline].
58.
Stalmans, W,
Bollen M,
and
Mvumbi L.
Control of glycogen synthesis in health and disease.
Diabetes Metab Rev
3:
127-161,
1987[Medline].
59.
Sun, AM,
Liu Y,
Dworkin LD,
Tse CM,
Donowitz M,
and
Yip KP.
Na+/H+ exchanger isoform 2 (NHE2) is expressed in the apical membrane of the medullary thick ascending limb.
J Membr Biol
160:
85-90,
1997[ISI][Medline].
60.
Takahashi, N,
Ito O,
and
Abe K.
Tubular effects of insulin.
Hypertens Res
19, Suppl1:
S41-S45,
1996[Medline].
61.
Talor, Z,
Emmanouel DS,
and
Katz AI.
Insulin binding and degradation by luminal and basolateral tubular membranes from rabbit kidney.
J Clin Invest
69:
1136-1146,
1982[ISI][Medline].
62.
Wang, T,
Yang CL,
Abbiati T,
Schultheis PJ,
Shull GE,
Giebisch G,
and
Aronson PS.
Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice.
Am J Physiol Renal Physiol
277:
F298-F302,
1999
63.
Wang, T,
Yang CL,
Abbiati T,
Shull GE,
Giebisch G,
and
Aronson PS.
Essential role of NHE3 in facilitating formate-dependent NaCl absorption in the proximal tubule.
Am J Physiol Renal Physiol
281:
F288-F292,
2001
64.
Weinman, EJ,
Steplock D,
Donowitz M,
and
Shenolikar S.
NHERF associations with sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3.
Biochemistry
39:
6123-6129,
2000[ISI][Medline].
65.
Wiederkehr, MR,
Di Sole F,
Collazo R,
Quinones H,
Fan L,
Murer H,
Helmle-Kolb C,
and
Moe OW.
Characterization of acute inhibition of Na/H exchanger NHE-3 by dopamine in opossum kidney cells.
Kidney Int
59:
197-209,
2001[ISI][Medline].
66.
Wiederkehr, MR,
Zhao H,
and
Moe OW.
Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation.
Am J Physiol Cell Physiol
276:
C1205-C1217,
1999
67.
Wu, MS,
Biemesderfer D,
Giebisch G,
and
Aronson PS.
Role of NHE3 in mediating renal brush border Na+-H+ exchange. Adaptation to metabolic acidosis.
J Biol Chem
271:
32749-32752,
1996
68.
Yang, X,
Amemiya M,
Peng Y,
Moe OW,
Preisig PA,
and
Alpern RJ.
Acid incubation causes exocytic insertion of NHE3 in OKP cells.
Am J Physiol Cell Physiol
279:
C410-C419,
2000
69.
Yip, KP,
Tse CM,
McDonough AA,
and
Marsh DJ.
Redistribution of Na+/H+ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension.
Am J Physiol Renal Physiol
275:
F565-F575,
1998
70.
Zhang, Y,
Magyar CE,
Norian JM,
Holstein-Rathlou NH,
Mircheff AK,
and
McDonough AA.
Reversible effects of acute hypertension on proximal tubule sodium transporters.
Am J Physiol Cell Physiol
274:
C1090-C1100,
1998
71.
Zhang, Y,
Norian JM,
Magyar CE,
Holstein-Rathlou NH,
Mircheff AK,
and
McDonough AA.
In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition.
Am J Physiol Renal Physiol
276:
F711-F719,
1999
72.
Zhao, H,
Wiederkehr MR,
Fan L,
Collazo RL,
Crowder LA,
and
Moe OW.
Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase A and NHE-3 phosphoserines 552 and 605.
J Biol Chem
274:
3978-3987,
1999
73.
Zizak, M,
Lamprecht G,
Steplock D,
Tariq N,
Shenolikar S,
Donowitz M,
Yun CH,
and
Weinman EJ.
cAMP-induced phosphorylation and inhibition of Na+/H+ exchanger 3 (Nhe3) are dependent on the presence but not the phosphorylation of Nhe regulatory factor.
J Biol Chem
274:
24753-24758,
1999