Role of NHE isoforms in mediating bicarbonate reabsorption along the nephron

Tong Wang1, Max Hropot2, Peter S. Aronson1,3, and Gerhard Giebisch1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> (JHCO3) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced Jv and JHCO3, but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, JHCO3 was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, JHCO3 was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na+/H+ exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.

sodium/hydrogen exchange; acidification; proximal; loop of Henle; distal convoluted tubule; sodium/hydrogen exchanger


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption mediated by Na+/H+ exchange in the proximal tubule (11, 17, 26, 39). However, the relative contributions of NHE3 and NHE2 in other nephron segments have not been fully evaluated.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the proximal tubule is S-3226 sensitive and Hoe-694 resistant, which is consistent with previous work indicating a predominant role for NHE3. Similarly, we find that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the loop of Henle is S-3226 sensitive and Hoe-694 resistant, which indicates a possible role for NHE3. In contrast, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the distal convoluted tubule is Hoe-694 sensitive and S-3226 resistant, which indicates that NHE2 rather than NHE3 is responsible for apical Na+/H+ exchange in this nephron segment.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the proximal tubule were expressed as the absorption rate per millimeter of tubule length.

Microperfusion of the loops of Henle was performed by a method similar to that detailed earlier (38). A proximal tubule was microperfused to locate its last loop on the kidney surface. The loop of Henle was then microperfused at a rate of 20 nl/min through a micropipette placed in this loop beyond an oil block. Fluid was collected from the first segment of the early distal tubule of the same nephron through a micropipette inserted upstream of another oil block. Under this condition, the loop of Henle was functionally isolated between the proximal and distal oil blocks. The rates of fluid and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the loop of Henle were expressed as the absorption rate per loop.

Distal convoluted tubules (early surface distal tubules) were perfused at a rate of 12 nl/min through a micropipette inserted downstream of an oil block in the first surface loop (37). Distal tubule fluid was collected from the second or third segment on the kidney surface ahead of a second oil block. The rates of fluid and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the distal tubule were expressed as the absorption rate per millimeter of tubule length (as were determined for the proximal tubule).

Measurement of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and fluid absorption. After each fluid collection, the pipette was withdrawn from the tubule into the oil covering the surface of the kidney, and a small amount of oil was aspirated into the tip of the collection pipette to prevent evaporation of the sample. The rates of absorption of fluid (JV) and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (JHCO3) were determined from the changes in [3H]methoxy-inulin activity and the total CO2 concentration between the perfusion solution and the collected fluid. A constant-bore glass capillary was used to take precise aliquots of initial perfusates and thereby collect samples that were analyzed for [3H]methoxy-inulin by liquid scintillation spectroscopy. The total CO2 concentration was measured by a microcalorimetric method (Picapnotherm), and the net JV and JHCO3 values were calculated as described previously (34, 36). The composition of the perfusion fluid used for all segments was as follows (in mM): 115 NaCl, 25 NaHCO3, 4 KCl, 1 CaCl2, 5 sodium acetate, 2.5 Na2HPO4, 0.5 NaH2PO4, 5 L-alanine, and 5 glucose. Solutions were bubbled at room temperature with a 5% CO2-95% O2 gas mixture before use. The pH was adjusted to 7.4 with a small amount of NaOH or HCl. The perfusion solution also contained 20 µCi/ml low-sodium [3H]methoxy-inulin (New England Nuclear, Boston, MA) for measuring volume absorption and 0.1% FD & C green dye for identification of the perfused loops.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Effects of ethylisopropylamiloride, Hoe-694, and S-3226 on fluid and bicarbonate absorption in proximal tubule of rat kidney



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Fig. 1.   Effects of inhibitors ethylisopropylamiloride (EIPA), Hoe-694, and S-3226 on rates of absorption of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (JHCO3) and fluid (Jv) in microperfused proximal tubules. Values are means ± SE; *P < 0.05 vs. control.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> and fluid absorption in the loop of Henle, which is defined as the portion of the nephron between the last surface loop of the proximal tubule and the first surface loop of the distal tubule. This portion of the nephron includes the pars recta, the descending and ascending limbs of the loop of Henle, and a short portion of the distal convoluted tubule. Shown in Table 2 and Fig. 2, JHCO3 and Jv in the loop of Henle were not significantly inhibited by 4 µM S-3226 or by 100 µM Hoe-694. Luminal concentrations of amiloride analogs in microperfused tubules are known to decline due to the appreciable permeability to these drugs (22). Accordingly, it is possible that the lack of effect of 4 µM S-3226 may have been due to the loss of the drug from the lumen of the loop segments. We therefore tested a higher concentration of S-3226 (40 µM) and observed significant inhibition of JHCO3 and Jv in the loop of Henle (as shown in Table 2 and Fig. 2). As in the proximal tubule, these results are consistent with the conclusion that NHE3 contributes significantly to apical membrane Na+/H+ exchange in the loop of Henle.

                              
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Table 2.   Effects of Hoe-694 and S-3226 on fluid and bicarbonate absorption in loop of Henle of rat kidney



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Fig. 2.   Effects of inhibitors on JHCO3 and Jv in microperfused loops of Henle. Values are means ± SE; *P < 0.05 vs. control.

We also examined the relative roles of NHE3 and NHE2 in mediating HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and fluid absorption in the early distal tubule (as summarized in Table 3 and Fig. 3). In contrast to transport in the other nephron segments that we studied, JHCO3 and Jv in the distal tubule were significantly inhibited by 100 µM Hoe-694. Moreover, transport in the distal tubule was unaffected by 1 µM and 40 µM S-3226, which are concentrations that significantly inhibited JHCO3 and Jv in the proximal tubule and loop of Henle, respectively. These findings strongly suggest that NHE2 rather than NHE3 accounts for apical membrane Na+/H+ exchange in the distal convoluted tubule.

                              
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Table 3.   Effects of Hoe-694 and S-3226 on fluid and bicarbonate absorption in distal tubule of rat kidney



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Fig. 3.   Effects of inhibitors on JHCO3 and Jv in microperfused distal tubules. Values are means ± SE. *P < 0.05 vs. control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

The general goal of the present study was to evaluate the relative roles of NHE3 and NHE2 in mediating HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in different segments of the nephron. Our experimental approach was to compare sensitivity of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in microperfused nephron segments to inhibition by Hoe-694 and S-3226. Our principal findings were that whereas NHE3 rather than NHE2 mediates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the proximal tubule and possibly in the loop of Henle, NHE2 rather than NHE3 is responsible for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in the distal convoluted tubule.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the proximal tubule (11, 39). The present study adds further evidence in support of the predominant role of NHE3 by demonstrating the sensitivity of proximal tubule HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption to the relatively selective NHE3 inhibitor S-3226. Similar findings have been recently reported by another group (32). However, it should be noted that a component of EIPA-sensitive proton secretion from proximal tubule cells can be detected in NHE3 null mice, which raises the possibility that another NHE isoform or a different EIPA-sensitive process contributes to proximal acidification (11).

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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption was sensitive to inhibition by 40 µM S-3226 but not by 100 µM Hoe-694, which is consistent with the functional contribution of NHE3 rather than NHE2.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the loop of Henle than in the proximal tubule. One likely explanation is that because the length of the perfused loop segment exceeds that of the perfused proximal tubule, there is greater loss of inhibitor from the lumen. Thus a higher concentration of S-3226 is needed to inhibit NHE3 in the loop than in the proximal tubule. Alternatively, one might consider the possibility that the 40 µM concentration of S-3226 used in our loop perfusions inhibited NHE2. However, this possibility is very unlikely because NHE2 is known to be >10-fold more sensitive to inhibition by Hoe-694 [I50 = 5 µM (12)] compared with S-3226 [I50 = 80 µM (27)], and we observed no inhibition of loop HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and fluid absorption by 100 µM Hoe-694. It should be noted that our data are not sufficient to prove that NHE3 accounts for apical Na+/H+ exchange in the loop of Henle. Although loss of S-3226 from the lumens of the long perfused segments provides a plausible explanation for why a higher concentration of the inhibitor would be required to inhibit NHE3 in the loop than in the proximal tubule, we cannot exclude the possibility that an NHE isoform other than NHE3 or NHE2 is present and contributes to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the loop of Henle.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption was not sensitive to inhibition by S-3226 but was inhibited by Hoe-694, which is consistent with the functional contribution of NHE2 rather than NHE3. Whether NHE2 accounts for all apical Na+/H+ exchange in the distal tubule was not assessed.

It is possible that the expression of specific apical NHE isoforms in different nephron segments allows differential regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. Indeed, recent evidence indicates that NHE2 and NHE3 differ in their responses to elevation of intracellular calcium or activation of protein kinases A or C (5, 15, 16, 18). Future studies will need to address differences in signaling systems that regulate NHE3 in the proximal tubule and loop of Henle and NHE2 in the distal convoluted tubule.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> and aldosterone (25). These findings imply that the loss of NHE2-mediated Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the distal tubule can be easily compensated, which suggests that the absolute rate of NHE2-mediated NaHCO3 reabsorption in this nephron segment is modest under normal conditions. It remains to be investigated whether NHE2-mediated NaHCO3 reabsorption in the distal tubule plays a larger physiological role in acidosis or in states of increased NaHCO3 delivery to this segment.


    ACKNOWLEDGEMENTS

The authors are grateful to Dr. H. J. Lang for helpful suggestions in preparing the manuscript.


    FOOTNOTES

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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RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1117-F1122
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