1 Department of Cellular and Molecular Physiology; 2 Aventis Pharma Deutschland, Frankfurt am Main 65926, Germany; and 3 Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8026
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ABSTRACT |
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This study assessed the functional role of
Na+/H+ exchanger (NHE) isoforms NHE3 and NHE2
in the proximal tubule, loop of Henle, and distal convoluted tubule of
the rat kidney by comparing sensitivity of transport to inhibition by
Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an
agent with much higher affinity for NHE3 than NHE2). Rates of transport
of fluid (Jv) and HCO
sodium/hydrogen exchange; acidification; proximal; loop of Henle; distal convoluted tubule; sodium/hydrogen exchanger
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INTRODUCTION |
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BICARBONATE
REABSORPTION mediated by apical membrane
Na+/H+ exchange has been observed in the
proximal tubule, the thick ascending limb of the loop of Henle, and the
distal convoluted tubule (6, 7, 10, 13, 14, 22, 37). To
date, expression of four Na+/H+ exchanger (NHE)
isoforms in the mammalian kidney has been described (19, 24, 30,
31). Immunocytochemical studies have indicated that NHE1 and
NHE4 have a basolateral distribution (3, 8, 20, 21, 23),
whereas NHE2 and/or NHE3 are located along the apical membranes of
various nephron segments including the proximal tubule, loop of
Henle,and distal convoluted tubule (1, 2, 4, 9, 20, 29,
41). Studies in NHE3 and NHE2 null mice suggest that NHE3 rather
than NHE2 is responsible for almost all HCO
The availability of inhibitors with major differences in affinity for
NHE3 compared with NHE2 allows assessment of the relative contributions of these two isoforms to bicarbonate reabsorption in
various nephron segments. The purpose of the present study was to
assess the functional role of NHE isoforms by comparing sensitivity of
transport to inhibition by Hoe-694 (12), an agent known to
inhibit NHE1 and NHE2 but not NHE3, and to S-3226 (27), an
agent with much higher affinity for NHE3 than for NHE2. We find that
HCO
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METHODS |
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Animals and surgical preparation. Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 200-250 g were maintained on standard rat chow and tap water until the day of the experiment. Rats were anesthetized by intraperitoneal injection of 100 mg/kg body wt of 5-ethyl-5-(L-methylpropyl)-2-thiobarbituric acid (Inactin, BYK-Gulden, Konstanz, Germany) and placed on a thermostatically controlled surgical table to maintain body temperature at 37°C. Three to five animals were used in each experimental group. After a tracheotomy was performed, the left jugular vein was exposed and cannulated with a PE-10 catheter for infusion of 0.9% saline at a rate of 1.5 ml/h. A carotid artery was catheterized with PE-10 tubing for collection of blood and measurement of mean arterial pressure.
Microperfusion of renal tubules.
After surgical preparation of the rats was complete, the left kidney
was exposed, immobilized in a kidney cup filled with light mineral oil,
and illuminated with a fiber-optic light source. Microperfusion of
proximal convoluted tubules in vivo was performed as described
previously (34, 36). A proximal convoluted tubule with
3-5 loops on the kidney surface was selected and perfused at a
rate of 20 nl/min using a micropipette inserted downstream of an oil
block. Tubule-fluid collections were made through a micropipette
inserted upstream of a second oil block. Subsequently the perfused
tubules were marked with Sudan Black heavy mineral oil and later filled
with high-viscosity microfil (Canton Bio-Medical Products, Boulder,
CO). After the kidney was partially digested, silicone rubber casts of
the tubule segments were dissected to determine the lengths of the
perfused segments. The rates of fluid and HCO
Measurement of HCO
Materials. [3H]methoxy-inulin was obtained from New England Research Products (Boston, MA), and ethylisopropylamiloride (EIPA) was purchased from Research Biochemicals International. Hoe-694 and S-3226 were generously provided by Dr. H. J. Lang at Aventis Pharma Deutschland (Frankfurt am Main, Germany).
Statistics. Data are presented as means ± SE. Dunnett's tests were 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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Effects of NHE inhibitors on JHCO3 and
Jv in the proximal convoluted tubule of the rat
kidney are summarized in Table 1 and Fig.
1. JHCO3 and
Jv were both significantly inhibited by 100 µM
EIPA, which confirms previous results (22, 35). These
findings are consistent with an important role of apical membrane
Na+/H+ exchange in mediating proximal
NaHCO3 absorption.
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To confirm that NHE3 is the isoform responsible for apical membrane Na+/H+ exchange in the proximal tubule, we compared the sensitivity of transport to inhibition by Hoe-694 [an agent that is known to inhibit NHE2 but not NHE3 (12)] and S-3226 [an agent with much higher affinity for NHE3 than for NHE2 (27)]. As indicated in Table 1 and Fig. 1, whereas JHCO3 and Jv in the proximal tubule were both significantly reduced by the presence of 1 µM S-3226, there was no detectable effect of 100 µM Hoe-694 [a concentration predicted to inhibit NHE2 (12)]. These results are consistent with the conclusion that NHE3 rather than NHE2 mediates apical membrane Na+/H+ exchange in the proximal tubule.
Similar studies were performed to evaluate the relative roles of NHE3
and NHE2 in mediating HCO
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We also examined the relative roles of NHE3 and NHE2 in mediating
HCO
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DISCUSSION |
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The general goal of the present study was to evaluate the relative
roles of NHE3 and NHE2 in mediating HCO
These observations extend previous results on the relative roles of
NHE2 and NHE3 in mediating acid-base transport along the nephron. In
the proximal tubule, immunocytochemical analysis has indicated the
presence of NHE3 on the brush-border membrane (1, 2, 4).
Reported results concerning the expression of NHE2 in the proximal
tubule have been conflicting (9, 41).
Na+/H+ exchange in rat brush-border membrane
vesicles is Hoe-694 insensitive, which indicates little if any
contribution of NHE2 to transport in this experimental preparation
(40). Moreover, studies in mice with deletion of NHE genes
indicate that NHE3 but not NHE2 contributes significantly to
HCO
In the loop of Henle, immunocytochemical analysis has indicated the
expression of both NHE2 and NHE3 on the apical membranes of cortical
and medullary thick ascending limb cells (1, 4, 9, 29).
Both NHE3 and NHE2 have been detected in the thin limbs of the loop of
Henle as well (1, 4, 9). In the present study, we perfused
the loop of Henle from the last surface loop of a proximal tubule to
the first surface loop of the distal convoluted tubule. Our results are
thus confined to short loops of Henle in superficial cortical nephrons.
In these segments, we found that HCO
Our findings are in apparent conflict with recently reported results
indicating that Na+ and fluid absorption in the loop of
Henle of the rat similarly microperfused in vivo is insensitive to
S-3226 (32). One difference between the two studies is our
use of a higher S-3226 concentration (40 µM vs. 30 µM). Indeed, we
found that a higher concentration of S-3226 was required to inhibit
HCO
In the distal tubule, immunocytochemical analysis has indicated the
expression of NHE2 on the apical membrane of macula densa cells
(20), distal convoluted tubule cells (9), and
connecting tubule cells (9). NHE3 has not been detected by
immunocytochemistry in the distal tubule (1, 4) although
it was found to be expressed in the porcine distal tubule by in situ
hybridization (28). In the present study, we perfused the
first loops of the superficial distal tubule. Our results are thus
confined to the distal convoluted tubule and the early connecting
tubule. In these nephron segments, we found that HCO
It is possible that the expression of specific apical NHE isoforms in
different nephron segments allows differential regulation of
HCO
Finally, it should be noted that whereas NHE3 null mice have mild
metabolic acidosis and a marked increase in aldosterone that reflects a
state of volume depletion (26), NHE2 null mice have normal
plasma HCO
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. H. J. Lang for helpful suggestions in preparing the manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-33793 and DK-17433. Portions of the study were previously published in abstract form. (J Am Soc Nephrol 10: 10A, 1999).
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.
Received 14 April 2000; accepted in final form 10 July 2001.
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