Formate-stimulated NaCl absorption in the proximal tubule is independent of the pendrin protein

Lawrence P. Karniski1, Tong Wang2, Lorraine A. Everett3, Eric D. Green3, Gerhard Giebisch2, and Peter S. Aronson2,4

1 Department of Internal Medicine, Veterans Affairs Medical Center and University of Iowa College of Medicine, Iowa City, Iowa 52242; Departments of 2 Cellular and Molecular Physiology and 4 Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520; and 3 Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A significant fraction of active chloride reabsorption across the apical membrane of the proximal tubule is mediated by a chloride/formate exchange process, whereby intracellular formate drives the transport of chloride into the cell. When chloride/formate exchange operates in parallel with Na+/H+ exchange and H+-coupled recycling of formate, the net result is electroneutral NaCl reabsorption. Pendrin is the protein product of the PDS gene (SLC26A4) and functions in several different anion exchange modes, including chloride/formate exchange. Pendrin is expressed in the kidney and may serve as the transporter responsible for formate-dependent NaCl reabsorption. In the present study, Pds-knockout mice were used to determine the role of pendrin in proximal tubule chloride reabsorption. We show that formate-dependent NaCl absorption in microperfused proximal tubules is similar between wild-type and pendrin-deficient mice. In addition, there is no difference in the rate of formate-mediated chloride transport in brush-border membrane vesicles isolated from wild-type and pendrin-deficient mice. These studies demonstrate that pendrin is not responsible for formate-dependent NaCl reabsorption in the proximal tubule.

chloride/formate exchange; Pendred syndrome; PDS; SLC26A4


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN THE KIDNEY, A SIGNIFICANT portion of active chloride reabsorption across the proximal tubule apical membrane is mediated by chloride/formate exchange (23, 27, 30, 31, 33). This process, in parallel with Na+/H+ exchange and H+-coupled recycling of formate, results in net NaCl reabsorption (4, 14, 22). The protein responsible for chloride/formate exchange across the apical membrane of the proximal tubule has not been clearly identified.

A candidate for the proximal tubule chloride/formate exchanger is pendrin, which is encoded by the PDS gene and is a member of the SLC26 family of anion exchange proteins (9, 10). Pendrin is capable of functioning in the chloride/formate, chloride/bicarbonate, and chloride/iodide exchange modes (24, 26, 28). Pendrin does not accept oxalate as a substrate, and it has an inhibitor profile similar to that described for the proximal tubule chloride/formate exchanger (15, 24, 26). In the kidney, pendrin has been immunolocalized to the apical membrane of non-alpha -intercalated cells of the cortical collecting duct, where it is involved in the secretion of bicarbonate (21). Pendrin has not been detected in the proximal tubule by immunocytochemical methods (16, 21); however, Soleimani et al. (28), using both RT-PCR analysis of proximal tubule segments and immunoblot analysis of isolated microvillus membrane vesicles, reported that pendrin is expressed in the proximal tubule. This raises the possibility that pendrin is the protein responsible for chloride/formate exchange in this nephron segment.

To determine the role of pendrin in proximal tubule chloride reabsorption, we compared chloride/formate exchange activity in the proximal tubules of wild-type and pendrin-deficient mice. These studies demonstrate that pendrin is not the protein responsible for mediating proximal tubule chloride/formate exchange.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pds-knockout mice. Wild-type (Pds+/+) and Pds-knockout mice (Pds-/-) were obtained from matings of Pds+/- mice. The method of generation and the phenotypic characterization of the Pds-/- mice have been described previously (8). In each experiment, Pds+/+ and Pds-/- mice were matched for age and sex.

Microperfusion of proximal tubules in situ. Microperfusion of proximal tubules was performed as described previously (32). In brief, proximal convoluted tubules were perfused at a rate of 15 nl/min with a solution containing (in mM) 140 NaCl, 5.0 NaHCO3, 4.0 KCl, 2.0 CaCl2, 1.0 MgSO4, 1.0 dibasic sodium phosphate, and 1.0 monobasic sodium phosphate, pH 6.7. Formate was added as the sodium salt. In a previous study that examined formate-stimulated volume reabsorption in the rat proximal tubule (30), there was a significant increase in the rate of net fluid absorption (Jv) observed with either 50 or 500 µM formate, with little difference in volume reabsorption noted between these two concentrations. To be certain that formate-stimulated Jv is maximized in the mouse, we used 500 µM formate in the present study. For measuring volume absorption, 20 µCi/ml of low-sodium [3H]methoxyinulin was added to the perfusion solution. 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 dissection of silicone rubber casts. Calculation of Jv was based on changes in the concentrations of [3H]inulin, and the rates are expressed per millimeter tubule length. Data are presented as means ± SE. Experimental groups were compared with a control group by using Dunnett's test. Differences were considered significant if P < 0.05.

Isolation of brush-border membrane vesicles. For each experiment, brush-border membrane vesicles were isolated from pooled renal cortices of six to eight mice using the magnesium-aggregation method (3, 15). The following modifications were made for isolating brush-border membranes from mouse kidneys: the first low-speed centrifugation step was performed for 8 min at 2,320 g, the second low-speed centrifugation step for 8 min at 3,770 g, and the third low-speed centrifugation step for 8 min at 4,380 g in a Sorval SM-24 rotor. For each experiment, the membrane vesicle preparations for wild-type and Pds-knockout mice were performed simultaneously. The purified membranes were stored at 3-4°C and used within 24 h of preparation. Protein concentration was determined by the method of Lowry et al. (18), as modified by Peterson (20).

To measure formate-stimulated chloride uptake, the vesicles were preequilibrated for 2-3 h at 20°C in 113 mM mannitol, 60 mM HEPES, 30 mM potassium hydroxide, and 100 mM potassium gluconate, pH 7.5, or with isosmotic replacement of potassium gluconate by 30 mM potassium formate. The uptake of 3.2 mM [36Cl]NaCl was assayed in the presence of 112 mM mannitol, 59 mM HEPES, 30 mM potassium hydroxide, and 100 mM potassium gluconate, pH 7.5, or with isosmotic replacement of potassium gluconate by 3 mM potassium formate. Valinomycin in ethanol was added to the membranes (20-30 µg/mg protein) in the presence of equimolar concentrations of potassium to prevent the development of a membrane potential generated by the imposed formate gradient. The formate-stimulated component of chloride uptake was determined by subtracting the rate of 36Cl uptake in the absence of intravesicular formate from the rate of 36Cl uptake in the presence of formate. For each experimental condition, the 20-s uptake of chloride was performed in triplicate, and the data are presented as the means ± SE of five experiments using different membrane preparations for each experiment. Statistical significance was determined using the paired Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To test whether pendrin is involved in formate-mediated chloride reabsorption in the proximal tubule, we first compared the effect of formate on fluid absorption in perfused proximal tubules from age- and sex-matched wild-type and Pds-knockout mice. Proximal tubules were microperfused in situ with a solution similar to that found in the late proximal tubule (high-chloride, low-bicarbonate, pH 6.7). Under these conditions, Jv reflects net NaCl absorption. If pendrin is the protein responsible for chloride/formate exchange in the proximal tubule, we would expect a significant reduction in formate-stimulated Jv in proximal tubules from pendrin-deficient mice compared with wild-type mice.

As shown in Table 1 and Fig. 1, volume absorption in the absence of formate was similar between wild-type and Pds-knockout mice. Furthermore, the addition of 500 µM formate to the luminal perfusate stimulated the rate of fluid absorption in both groups of mice. These results are consistent with luminal membrane chloride/formate exchange mediating transcellular NaCl reabsorption, as previously demonstrated in rats, rabbits, and mice (23, 27, 31-33). There was no significant difference between the formate-stimulated component of fluid absorption between wild-type and Pds-knockout mice, suggesting that pendrin is not the protein responsible for chloride/formate exchange in the proximal tubule.

                              
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Table 1.   Effect of formate on fluid absorption in proximal tubules of wild-type and knockout mice



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Fig. 1.   Proximal tubule fluid absorption (Jv) in wild-type (Pds+/+) and pendrin-deficient (Pds-/-) mice in the presence (filled bars) or absence (open bars) of 500 µM formate.

To directly compare the rates of chloride/formate exchange in wild-type and Pds-knockout mice, we measured formate-dependent chloride uptake in brush-border membrane vesicles. As illustrated in Fig. 2, the purification of the brush-border membrane marker gamma -GTP was not significantly different in membrane preparations obtained from wild-type and Pds-knockout mice (10.8- and 11.6-fold purification, respectively). This demonstrates that the purification of the brush-border membrane preparations is similar between the two groups of mice. The uptake of 36Cl was subsequently measured in isolated membrane vesicles in the presence and absence of an outwardly directed formate gradient (Fig. 3). We were unable to detect a significant difference in formate-stimulated chloride transport between wild-type and pendrin-deficient mice. This result is consistent with the results obtained in the perfused proximal tubules and confirms that pendrin is not responsible for significant chloride/formate exchange across the apical membrane of the proximal tubule cell.


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Fig. 2.   The purity of membrane vesicles from Pds+/+ and Pds-/- mice is expressed as the ratio of the specific activity of gamma -GTP in brush-border membrane vesicles (BBMV) to the specific activity of gamma -GTP in whole homogenates. P > 0.4 between the 2 groups.



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Fig. 3.   Chloride/formate exchange in brush-border membrane vesicles from Pds+/+ and Pds-/- mice. The uptake of 36Cl was measured in brush-border membrane vesicles in the presence (filled bars) or absence (open bars) of an outwardly directed formate gradient. P < 0.01 within each group (Pds+/+ and Pds-/-) between gluconate controls and formate.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pendrin is the protein product of the PDS gene and functions as an anion exchanger, accepting chloride, iodide, bicarbonate, and formate as substrates (9, 24, 26, 28). Individuals homozygous for disease-causing mutations of the human PDS gene present with hearing loss associated with malformations of the inner ear (12, 13, 17, 29). Deafness is thought to result from an inability to transport chloride in the cochlea and endolymphatic duct, where pendrin is expressed. Individuals with PDS mutations that result in complete loss of pendrin function may also develop Pendred syndrome, presenting with thyroid abnormalities in addition to hearing loss (19, 25). The thyroid disorders are manifested by an incomplete organification of thyroglobulin, resulting from defective pendrin-mediated iodide transport across the apical membrane of thyrocytes.

In addition to its expression in the inner ear and thyroid, pendrin is also expressed in the kidney (9). We have previously detected pendrin on the apical membrane of a subpopulation of intercalated cells and determined that pendrin functions as a mechanism of bicarbonate secretion in the cortical collecting duct of the mouse (21). The exact intercalated cell type in which pendrin is located is unclear; however, the fact that there is no overlap between cells staining for pendrin and anion exchanger 1 (AE1) suggests that pendrin is not located on alpha -intercalated cells.

In the proximal tubule, chloride/formate exchange performs a significant role in NaCl reabsorption (23, 27, 30, 31, 33). The functional similarity between pendrin and the proximal tubule chloride/formate exchanger makes pendrin a likely candidate as the protein responsible for formate-mediated chloride reabsorption in the proximal tubule. Three different studies have been unable to immunolocalize pendrin to the proximal tubule using immunocytochemical techniques (16, 21, 28), although pendrin has been found on the apical membrane of the cortical collecting duct in rats, humans, and mice (16, 21). The inability to immunolocalize pendrin in the proximal tubule suggests either that pendrin is not expressed in this nephron segment or that the pendrin protein in the proximal tubule is inaccessible to the immunocytochemical probes. In contrast, pendrin has been identified in the proximal tubule using immunoblots of membrane vesicles and RT-PCR of individually dissected proximal tubule segments (28). Interestingly, the expression of pendrin by RT-PCR is significantly less in the proximal tubule compared with the cortical collecting duct (28), despite the fact that pendrin is present in only a small, subpopulation of cortical collecting duct cells (21). This suggests that the level of pendrin expression in the proximal tubule may be too low to be functionally significant.

In the present study, we used Pds-knockout mice to determine whether pendrin is the protein responsible for chloride/formate exchange in the proximal tubule. In the absence of formate, volume absorption was detected in both wild-type and Pds-knockout mice, consistent with previous findings in rats, rabbits, and mice (23, 31-33). A significant portion of the baseline volume absorption represents passive chloride transport through paracellular pathways (2, 4, 7, 11). In addition, in the absence of other organic acids such as formate, it has been proposed that either chloride/bicarbonate or chloride/hydroxyl exchange acting in parallel with Na+/H+ exchange provides a mechanism of NaCl reabsorption in the proximal tubule, although this process probably contributes very little to overall transcellular NaCl transport (1, 5, 6, 31).

When we added formate to the luminal perfusate, a significant increase in volume absorption was observed; however, we were unable to detect a difference between wild-type and Pds-knockout mice in either the rate of formate-mediated volume reabsorption in perfused proximal tubules or the rate of chloride/formate exchange in isolated brush-border membrane vesicles. These studies demonstrate that pendrin is not the protein responsible for proximal tubule chloride/formate exchange.

The fact that pendrin-deficient mice have normal rates of chloride/formate exchange conflicts with the report that pendrin is expressed in the proximal tubule (28). One possible explanation is that pendrin does not function as a chloride/formate exchanger in the proximal tubule but instead mediates either chloride/hydroxyl or chloride/bicarbonate exchange. This would seem unlikely for two reasons. First, our studies in perfused proximal tubules did not demonstrate any difference in Jv between wild-type and Pds-knockout mice in the absence of formate, despite the luminal acid-pH gradient imposed across the apical membrane. If pendrin mediates significant chloride/bicarbonate exchange in parallel with apical membrane Na+/H+ exchange, we would have expected to see a reduction in Jv in pendrin-deficient mice under these conditions. Second, if pendrin mediates significant chloride reabsorption by either chloride/formate exchange or chloride/bicarbonate exchange, we should have detected a difference in Jv between wild-type and Pds-knockout mice after the addition of formate, because formate is preferred over bicarbonate as a substrate for pendrin (28).

In conclusion, we find that proximal tubules of Pds-knockout mice have rates of formate-mediated volume absorption and chloride/formate exchange similar to those in control mice. This suggests that the level of pendrin expression in the proximal tubule is insufficient to mediate significant NaCl reabsorption in this nephron segment.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-17433 (to T. Wang, G. Giebisch, and P. S. Aronson) and DK-47881, the March of Dimes Birth Foundation, and the Office of Research and Development, Department of Veterans Affairs (to L. P. Karniski).


    FOOTNOTES

Address for reprint requests and other correspondence: L. P. Karniski, Dept. of Internal Medicine, Univ. of Iowa Hospitals, 200 Hawkins Dr., Iowa City, IA 52242 (E-mail: lawrence-karniski{at}uiowa.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.

10.1152/ajprenal.00182.2002

Received 9 May 2002; accepted in final form 27 June 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 283(5):F952-F956