Essential role of NHE3 in facilitating formate-dependent NaCl absorption in the proximal tubule

Tong Wang1, Chao-Ling Yang2, Thecla Abbiati2, Gary E. Shull3, Gerhard Giebisch1, and Peter S. Aronson1,2

Departments of 1 Cellular and Molecular Physiology, and 2 Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8029; and 3 Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The absorption of NaCl in the proximal tubule is markedly stimulated by formate. This stimulation of NaCl transport is consistent with a cell model involving Cl--formate exchange in parallel with pH-coupled formate recycling due to nonionic diffusion of formic acid or H+-formate cotransport. The formate recycling process requires H+ secretion. Although Na+-H+ exchanger isoform NHE3 accounts for the largest component of H+ secretion in the proximal tubule, 40-50% of the rates of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption or cellular H+ extrusion persist in NHE3 null mice. The purpose of the present investigation is to use NHE3 null mice to directly test the role of apical membrane NHE3 in mediating NaCl absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, but the component of NaCl absorption stimulated by formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, indicating that oxalate-stimulated NaCl absorption is independent of Na+-H+ exchange. The virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.

oxalate; anion exchange; Na+-H+ exchanger; microperfusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ABSORPTION OF NaCl in the proximal tubule is markedly stimulated by formate and oxalate (13, 20, 22, 24, 25, 27). This stimulation of NaCl transport is consistent with a cell model involving Cl--formate and Cl--oxalate exchange processes in parallel with recycling of formate and oxalate across the apical membrane (3). In the case of formate-stimulated NaCl absorption, it has been proposed that there is pH-coupled formate recycling due to nonionic diffusion of formic acid or H+-formate cotransport (10, 19). Such a process in turn requires H+ secretion in parallel with Cl--formate exchange. In the proximal tubule, Na+-H+ exchanger isoform NHE3 plays a major role in H+ secretion, but 40-50% of the rates of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption or cellular H+ extrusion persist in NHE3 null mice (7, 21, 26).

The purpose of the present investigation is to use NHE3 null mice to directly test the role of apical membrane NHE3 in mediating NaCl absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, as previously found in the rat (24, 25, 27). Moreover, we find that the component of NaCl absorption stimulated by formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, consistent with the previous proposal that oxalate-stimulated NaCl absorption is independent of Na+-H+ exchange, and is mediated by Na+-sulfate cotransport in parallel with Cl--oxalate exchange and sulfate-oxalate exchange (3, 12, 24).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and surgical preparation. Knockout mice deficient in NHE3 were generated by targeted gene disruption (21). Genotype analysis of tail DNA was performed by PCR, using primers derived from the 5' and 3' ends of exon 6 and the 5' end of the neomycin resistance gene. When used in the same reaction, the three primers amplify a 199-bp product from the wild-type gene and a 113-bp product from the mutant gene. Homozygous wild-type (NHE+/+) and null (NHE3-/-) mice resulting from breeding of heterozygotes were maintained on a regular diet and tap water until the day of the experiment. Ages of mutant animals were matched with their wild-type controls.

During the course of our studies, heterozygotes were progressively back-crossed with Black Swiss mice. Heterozygotes used to generate the wild-type and null mice used in the present experiments were derived from two to four generations of back-crossing with this outbred strain. Wild-type and null mice were matched with respect to genetic background.

Microperfusion of proximal tubules in situ. The details of the methods for surgical preparation and microperfusion of mouse proximal tubules in vivo were described previously (21, 26) and were similar to those used in the rat (24). Briefly, proximal convoluted tubules with 3-5 loops on the kidney surface were perfused at a rate of 15 nl/min with a perfusion solution containing 20 µCi/ml of low-sodium [3H]methoxy-inulin for measuring volume absorption. Tubule fluid collections were made downstream with another micropipette. One collection was made in each perfused tubule, and two to four collections were taken in the experimental kidney of each animal. The perfused segments were marked with Sudan Black heavy mineral oil, and their lengths were determined after filling with high-viscosity microfil (Canton Bio-Medical Products, Boulder, Colorado) and dissection of the silicone rubber casts.

Measurement of rates of Cl- and fluid absorption. The rates of net Cl- (JCl) and fluid (Jv) absorption were calculated based on changes in the concentrations of [3H]inulin and Cl- as described previously (23, 24). JCl and Jv were expressed per millimeter tubule length. The composition of the perfusion solution was the same as used previously in the rat (24) (in mM): sodium chloride 140, sodium bicarbonate 5.0, potassium chloride 4.0, calcium chloride 2.0, magnesium sulfate 1.0, dibasic sodium phosphate 1.0, and monobasic sodium phosphate 1.0, pH 6.7. Concentrations of formate and oxalate indicated in the tables and figures were added as sodium salts.

Statistics. Data are presented as means ± SE. Experimental groups were compared with a control group by use of Dunnett's test. Differences were considered significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the first series of experiments, we evaluated whether formate and oxalate stimulate NaCl transport in the proximal tubule of the mouse as previously observed in the rat (24, 27). To this end, proximal tubules were microperfused in situ with a relatively high Cl-, low HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, pH 6.7 solution mimicking conditions in the late proximal tubule. Under these conditions, the rate of volume reabsorption, Jv, results predominantly from net NaCl absorption. The rate of Cl- reabsorption, JCl, was also measured directly.

Shown in Table 1 and Fig. 1, the addition of either 500 µM formate or 5 µM oxalate to the luminal perfusion solution resulted in significant stimulation of Jv and JCl. These findings are consistent with the contributions of luminal membrane Cl--formate and Cl--oxalate exchange processes to transcellular NaCl absorption as previously demonstrated in the rabbit and rat proximal tubule (13, 20, 22, 24, 25, 27). A key finding in the previous studies in the rat was that stimulation of Jv and JCl by formate but not by oxalate was abolished by luminal application of the Na+-H+ exchange inhibitor ethylisopropylamiloride (EIPA) (24). This finding strongly suggested a central role for apical membrane Na+-H+ exchange in mediating formate-stimulated NaCl absorption.

                              
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Table 1.   Effects of formate and oxalate on fluid and chloride absorption in wild-type mice



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Fig. 1.   Effects of formate (500 µM) and oxalate (5 µM) on rates of chloride (JCl) and fluid (Jv) absorption in wild-type mice.

Given that NHE3 is the principal Na+-H+ exchanger isoform in the brush border membrane of proximal tubule cells (2, 4, 14, 21, 29), it would seem most likely that inhibition of this isoform was responsible for the observed inhibition of formate-stimulated NaCl absorption by EIPA. However, an EIPA-sensitive component of apical membrane acid extrusion from proximal tubule cells was recently observed in isolated tubules from NHE3 null mice (7), suggesting that an Na+-H+ exchanger isoform other than NHE3 might be functional at the brush border membrane.

Accordingly, to directly address the specific role of NHE3 in formate-dependent NaCl transport, we assessed the ability of formate to stimulate NaCl transport in the proximal tubules of NHE3 null mice. These results are shown in Table 2 and Fig. 2. The baseline Jv and JCl measured in the absence of formate and oxalate were lower in the NHE3 null mice compared with the wild-type controls (see Table 1 and Fig. 1). Formate failed to increase Jv or JCl significantly in the proximal tubules of NHE3 null mice. In contrast, significant stimulation of Jv and JCl by oxalate was still present. The latter finding underscores the specific role of NHE3 in facilitating formate stimulation of NaCl transport in the proximal tubule.

                              
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Table 2.   Effects of formate and oxalate on fluid and chloride absorption in NHE3 null mice



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Fig. 2.   Effects of formate (500 µM) and oxalate (5 µM) on JCl and Jv absorption in Na+-H+ exchanger isoform NHE3 null mice.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A Cl--formate exchange activity was originally identified in studies of isolated brush border membrane vesicles (9, 10). For this anion exchange process to support significant absorptive Cl- flux, a mechanism is required to recycle formate back from the lumen into the cell in view of the low plasma concentrations of formate (13, 24). At least two potential mechanisms for formate recycling coupled to H+ translocation were identified in studies of brush border membrane vesicles, nonionic diffusion of formic acid, and H+-formate cotransport (or OH--formate exchange) (10, 19). These mechanisms in turn require continuous extrusion of H+ across the apical membrane. In the proximal tubule, the major pathway for cellular acid extrusion is Na+-H+ exchange (1, 11). Immunocytochemical studies have identified the expression of Na+-H+ exchanger isoform NHE3 on the brush border membrane (2, 4, 5), and the profile of inhibitor sensitivity of brush border Na+-H+ exchange is characteristic of NHE3 (29). The rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is reduced by ~50-60% in NHE3 null mice, further supporting the role of this isoform in mediating brush border acid secretion (21, 26). Of the remaining component of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in NHE3 null mice, ~60% is inhibited by bafilomycin, an inhibitor of the vacuolar H+-ATPase (26). Moreover, studies in isolated proximal tubules from NHE3/NHE2 null mice have identified a novel EIPA-sensitive acid extrusion process (7). This process might contribute to the net rates of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (JHCO3) observed to persist in NHE3 null mice in vivo in the presence of bafilomycin (26).

The models in Fig. 3 illustrate formate-dependent NaCl absorption as occurring by Cl--formate exchange in parallel with H+ secretion by either Na+-H+ exchange or H+-ATPase. As shown in Fig. 3A, in which H+ secretion takes place by Na+-H+ exchange, the Na+ absorption associated with formate-dependent Cl- transport is transcellular. In contrast, as shown in Fig. 3B, in which H+ secretion is mediated by H+-ATPase, Cl- absorption is electrogenic, and is accompanied by paracellular Na+ absorption. In either case, formate would induce net NaCl absorption.


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Fig. 3.   Models for formate-dependent NaCl absorption. A: NaCl absorption takes place by Cl--formate exchange with pH-coupled formate recycling and H+ secretion by Na+-H+ exchange. B: NaCl absorption takes place by Cl--formate exchange with pH-coupled formate recycling and H+ secretion by H+-ATPase. Na+ absorption is paracellular driven by the lumen-positive potential difference.

In microperfused proximal tubules of rabbit and rat, formate markedly stimulates Jv and JCl (13, 20, 22, 24, 25, 27), consistent with the proposed role of Cl--formate exchange in mediating NaCl absorption according to both of the models in Fig. 3. Moreover, the ability of formate to stimulate proximal NaCl absorption is dependent on luminal acidification, consistent with the necessity for H+-coupled formate recycling (20). Despite the evidence that a significant fraction of proximal acidification is independent of NHE3 (7, 21, 26), the Na+-H+ exchange inhibitor EIPA completely abolishes formate stimulation of Jv and JCl (24), suggesting a key role for NHE3 or another EIPA-sensitive process.

We now find that the ability of formate to stimulate NaCl transport is virtually abolished in the proximal tubules of NHE3 null mice. This observation indicates that neither another EIPA-sensitive process nor the H+-ATPase can sustain the H+ secretion needed for appreciable Cl- absorption by Cl--formate exchange to occur. Given that NHE3 accounts for only 50-60% of H+ secretion across the brush border membrane and that there exist several alternative mechanisms for acid extrusion, the question arises as to why there is an absolute requirement for NHE3 activity for formate-stimulated NaCl absorption. Indeed, in our studies, the tubules were perfused with a low HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution (5 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, pH 6.7) simulating the maximal acidification achieved along the proximal tubule, so it is not evident why there should be any dependence of formate-stimulated NaCl absorption on the activity of acid secretion processes. Thus the present results raise the possibility that there is an intimate relationship between NHE3 and the formate transporters to permit direct transfer of H+ between NHE3 and the formate recycling pathways. Such a mechanism would be analogous to substrate tunneling or channeling in multi-subunit enzyme systems in which substrate is directly transferred from one site to another without diffusion through the bulk phase (15).

It may be noted that the baseline Jv and JCl measured in the absence of added formate or oxalate are lower in the NHE3 null mice compared with the wild-type controls. One possible explanation is that a component of NaCl absorption dependent on NHE3 activity is present under baseline conditions due to Cl--formate exchange resulting from the presence of formate in peritubular capillary blood. Alternatively, Cl--base exchange independent of formate (e.g., Cl--OH- exchange) might be present, and, in parallel with Na+-H+ exchange, contribute to NaCl absorption (13, 22, 28). In addition, glomerular filtration rate is chronically and markedly reduced in NHE3 null mice (14), possibly resulting in reduced cell size, membrane surface area, and transporter expression, the opposite of what results from glomerular hyperfiltration (6, 8, 16-18).

Significant stimulation of Jv and JCl by oxalate is still observed in NHE3 null mice, reflecting the presence of the component of transcellular Cl- absorption taking place by Cl--oxalate exchange. These findings are consistent with the previous observation that EIPA does not inhibit oxalate-stimulated NaCl transport (24). Taken together, the present and previous results indicate that NHE3 has no role in mediating oxalate-stimulated NaCl absorption. In fact, previous evidence strongly suggests that oxalate-stimulated NaCl absorption is mediated by Na+-sulfate cotransport in parallel with Cl--oxalate exchange and sulfate-oxalate exchange (3, 12, 24).

In conclusion, we find that NHE3 has a specific role in mediating formate-stimulated NaCl absorption in the proximal tubule. In view of the fact that other secretory pathways contribute to H+ secretion in the proximal tubule, the virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-17433, DK-33793, and DK-50594.


    FOOTNOTES

Address for reprint requests and other correspondence: P. S. Aronson, Dept. of Internal Medicine, Yale School of Medicine, 333 Cedar St., P.O. Box 208029, New Haven, CT 06520-8029

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 20 February 2001; accepted in final form 17 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alpern, RJ. Cell mechanisms of proximal tubule acidification. Physiol Rev 70: 79-114, 1990[Free Full Text].

2.   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].

3.   Aronson, PS, and Giebisch G. Mechanisms of chloride transport in the proximal tubule. Am J Physiol Renal Physiol 273: F179-F192, 1997[Abstract/Free Full Text].

4.   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[Abstract/Free Full Text].

5.   Biemesderfer, D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, and Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289-F299, 1997[Abstract/Free Full Text].

6.   Celsi, G, Larsson L, and Aperia A. Proximal tubular reabsorption and Na-K-ATPase activity in remnant kidney of young rats. Am J Physiol Renal Fluid Electrolyte Physiol 251: F588-F593, 1986[ISI][Medline].

7.   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[Abstract/Free Full Text].

8.   Fine, LG, and Bradley T. Adaptation of proximal tubular structure and function: insights into compensatory renal hypertrophy. Fed Proc 44: 2723-2727, 1985[ISI][Medline].

9.   Karniski, LP, and Aronson PS. Anion exchange pathways for Cl- transport in rabbit renal microvillus membranes. Am J Physiol Renal Fluid Electrolyte Physiol 253: F513-F521, 1987[Abstract/Free Full Text].

10.   Karniski, LP, and Aronson PS. Chloride/formate exchange with formic acid recycling: a mechanism of active chloride transport across epithelial membranes. Proc Natl Acad Sci USA 82: 6362-6365, 1985[Abstract].

11.   Krapf, R, and Alpern RJ. Cell pH and transepithelial H/HCO3 transport in the renal proximal tubule. J Membr Biol 131: 1-10, 1993[ISI][Medline].

12.   Kuo, SM, and Aronson PS. Pathways for oxalate transport in rabbit renal microvillus membrane vesicles. J Biol Chem 271: 15491-15497, 1996[Abstract/Free Full Text].

13.   Kurtz, I, Nagami G, Yanagawa N, Li L, Emmons C, and Lee I. Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule. J Clin Invest 94: 173-183, 1994[ISI][Medline].

14.   Lorenz, JN, Schultheis PJ, Traynor T, Shull GE, and Schnermann J. Micropuncture analysis of single-nephron function in NHE3-deficient mice. Am J Physiol Renal Physiol 277: F447-F453, 1999[Abstract/Free Full Text].

15.   Miles, EW, Rhee S, and Davies DR. The molecular basis of substrate channeling. J Biol Chem 274: 12193-12196, 1999[Free Full Text].

16.   Ohno, A, Beck FX, Pfaller W, Giebisch G, and Wang T. Effects of chronic hyperfiltration on proximal tubule bicarbonate transport and cell electrolytes. Kidney Int 48: 712-721, 1995[ISI][Medline].

17.   Pfaller, W, Seppi T, Ohno A, Giebisch G, and Beck FX. Quantitative morphology of renal cortical structures during compensatory hypertrophy. Exp Nephrol 6: 308-319, 1998[ISI][Medline].

18.   Preisig, PA, and Alpern RJ. Increased Na/H antiporter and Na/3HCO3 symporter activities in chronic hyperfiltration. A model of cell hypertrophy. J Gen Physiol 97: 195-217, 1991[Abstract].

19.   Saleh, AM, Rudnick H, and Aronson PS. Mechanism of H(+)-coupled formate transport in rabbit renal microvillus membranes. Am J Physiol Renal Fluid Electrolyte Physiol 271: F401-F407, 1996[Abstract/Free Full Text].

20.   Schild, L, Giebisch G, Karniski LP, and Aronson PS. Effect of formate on volume reabsorption in the rabbit proximal tubule. J Clin Invest 79: 32-38, 1987[ISI][Medline].

21.   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].

22.   Sheu, JN, Quigley R, and Baum M. Heterogeneity of chloride/base exchange in rabbit superficial and juxtamedullary proximal convoluted tubules. Am J Physiol Renal Fluid Electrolyte Physiol 268: F847-F853, 1995[Abstract/Free Full Text].

23.   Wang, T, Agulian SK, Giebisch G, and Aronson PS. Effects of formate and oxalate on chloride absorption in rat distal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 264: F730-F736, 1993[Abstract/Free Full Text].

24.   Wang, T, Egbert AL, Jr, Abbiati T, Aronson PS, and Giebisch G. Mechanisms of stimulation of proximal tubule chloride transport by formate and oxalate. Am J Physiol Renal Fluid Electrolyte Physiol 271: F446-F450, 1996[Abstract/Free Full Text].

25.   Wang, T, Giebisch G, and Aronson PS. Effects of formate and oxalate on volume absorption in rat proximal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 263: F37-F42, 1992[Abstract/Free Full Text].

26.   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[Abstract/Free Full Text].

27.   Wareing, M, and Green R. Effect of formate and oxalate on fluid reabsorption from the proximal convoluted tubule of the anaesthetized rat. J Physiol (Lond) 477: 347-354, 1994[Abstract].

28.   Warnock, DG, and Yee VJ. Chloride uptake by brush border membrane vesicles isolated from rabbit renal cortex. Coupling to proton gradients and K+ diffusion potentials. J Clin Invest 67: 103-115, 1981[ISI][Medline].

29.   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[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 281(2):F288-F292
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