Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice

Tong Wang1, Chao-Ling Yang2, Thecla Abbiati2, Patrick J. Schultheis3, Gary E. Shull3, Gerhard Giebisch1, and Peter S. Aronson1,2

1 Departments of 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


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NHE3 is the predominant isoform responsible for apical membrane Na+/H+ exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3-/- mouse with greatly reduced proximal tubule HCO-3 absorption compared with NHE3+/+ animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282-285, 1998). The purpose of the present study was to evaluate the role of other acidification mechanisms in mediating the remaining component of proximal tubule HCO-3 reabsorption in NHE3-/- mice. Proximal tubule transport was studied by in situ microperfusion. Net rates of HCO-3 (JHCO3) and fluid absorption (Jv) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 µM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition of JHCO3 and Jv in NHE3+/+ mice but failed to inhibit JHCO3 or Jv in NHE3-/- mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 µM bafilomycin caused a similar absolute decrement in JHCO3 in wild-type and NHE3 null mice, indicating equivalent rates of HCO-3 absorption mediated by H+-ATPase. Addition of 10 µM Sch-28080 did not reduce JHCO3 in either wild-type or NHE3 null mice, indicating lack of detectable H+-K+-ATPase activity in the proximal tubule. We conclude that, in the absence of NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes to mediating HCO-3 reabsorption in the proximal tubule. A significant component of HCO-3 reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H+-ATPase, but its activity is not significantly upregulated in NHE3 null mice.

sodium/proton exchange; proton-adenosinetriphosphatase; proton-potassium-adenosinetriphosphatase; acidification


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A MAJOR FRACTION of proximal tubule HCO-3 reabsorption is mediated by apical membrane Na+/H+ exchange (1, 4). Molecular cloning studies have shown that at least four Na+/H+ exchanger (NHE) isoforms are expressed in the mammalian kidney (23, 26, 30, 31). Immunocytochemical studies using isoform-specific antibodies have indicated that, whereas NHE1 and NHE4 have a basolateral distribution (6, 9, 24), NHE2 and NHE3 are located along the apical membranes of various nephron segments, including the proximal tubule (3, 5, 7, 10, 28, 29, 39, 40). Analysis of inhibitor sensitivity has suggested that virtually all of the measured Na+/H+ exchange activity in isolated renal cortical brush-border membrane vesicles is mediated by NHE3 (38).

Direct evidence for the functional importance of NHE3 is that proximal tubule HCO-3 reabsorption is reduced by ~60% in NHE3 null mice (27). However, the mechanisms accounting for the remaining component of HCO-3 reabsorption in the proximal tubules of NHE3 null mice are not known. Possible mechanisms include participation of another apical NHE isoform, such as NHE2, or of primary active H+ transport pathways, such as H+-ATPase and H+-K+-ATPase. The aims of the present study are to evaluate the relative contributions of these possible mechanisms to mediating HCO-3 reabsorption in the proximal tubules of NHE3 null mice.


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Animals and surgical preparation. Knockout mice deficient in NHE3 were generated by targeted gene disruption (27). Genotype analysis of tail DNA was performed by PCR. 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. The mice were anesthetized by intraperitoneal injection of 100 mg/kg body wt Inactin [5-ethyl-5-(L-methylpropyl)-2-thiobarbituric acid; BYK-Gulden, Konstanz, Germany] and were placed on a thermostatically controlled surgical table to maintain body temperature at 37°C. After tracheotomy, the left jugular vein was exposed and cannulated with a PE-10 catheter for intravenous infusion. A carotid artery was also catheterized with PE-10 tubing for arterial blood collection for blood gas analysis and for measurement of mean arterial pressure. Blood gas analysis was performed on freshly drawn blood by use of a Corning Blood Gas Analyzer.

Microperfusion of proximal tubules in situ. After surgical preparation, saline solution (0.9%) was infused at a rate of 0.15 ml/h (<FR><NU>1</NU><DE>10</DE></FR> of the infusion rate used in rat). The left kidney was exposed by lateral abdominal incision, carefully isolated, and immobilized in a special kidney cup filled with light mineral oil (37°C). The kidney surface was illuminated by a fiber optical light. The details of the method for microperfusion of proximal tubules in vivo were described previously for the rat (35). Briefly, a proximal convoluted tubule with three to five loops on the kidney surface was selected and perfused at a rate of 15 nl/min with a proximal oil block. The perfusion solution contained 20 µCi/ml of low-sodium [methoxy-3H]inulin for measuring volume absorption and 0.1% FD & C green dye for identification of the perfused loops. Tubule fluid collections were made downstream with another micropipette with distal oil block. One collection was made in each perfused tubule, and two to four collections were taken in each kidney. The perfused tubules were marked after collection with sudan black heavy mineral oil. To determine the length of the perfused segment, tubules were filled with high-viscosity microfil (Canton Bio-Medical Products, Boulder, CO), the kidney was partially digested in 20% NaOH, and silicone rubber casts of the tubule segments were dissected.

Measurement of net HCO-3 and fluid absorption. The rates of net HCO-3 (JHCO3) and fluid (Jv) absorption were calculated based on changes in the concentrations of [3H]inulin and total CO2 as described previously (35). The total CO2 concentrations in both initial and collected fluids were measured by a microcalorimetric (Picapnotherm) method (35). JHCO3 and Jv were expressed per millimeter tubule length. The composition of the perfusion solution was the same as used previously in the rat (36) (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 titrated to 7.4 with NaOH or HCl as required.

Statistics. Data are presented as means ± SE. Student's t-test was used when a single experimental group was compared with a control group (Table 1). Several experimental groups were compared with a control group (Table 2) by use of Dunnett's test. Differences were considered significant at P < 0.05. 

                              
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Table 1.   Acid-base status in wild-type and NHE3 null mice


                              
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Table 2.   Fluid and bicarbonate absorption in proximal tubules of wild-type and NHE3 null mice


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Blood gas analysis, as shown in Table 1, indicated that NHE3 null mice have a mild to moderate metabolic acidosis with reduction in arterial blood HCO-3 concentration from 25.7 to 20.4 mM and reduction of pH from 7.34 to 7.18. A moderate respiratory acidosis was also noted in both sets of animals (PCO2 50-55), most likely secondary to anesthesia. The mild metabolic acidosis in NHE3 null mice confirms previous results (27).

As indicated in Table 2 and Fig. 1, JHCO3 and Jv were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. These findings confirm previous results indicating a major role of NHE3 in mediating proximal tubule HCO-3 and fluid absorption (27). Clearly, a significant fraction of JHCO3 persists in the NHE3 null mice, indicating contributions from alternative acidification processes. We therefore used inhibitors to investigate the mechanisms mediating the remaining JHCO3 and Jv.


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Fig. 1.   Effects of inhibitors on rates of HCO-3 (JHCO3) and fluid (Jv) absorption in wild-type and NHE3 null mice. EIPA, ethylisopropylamiloride; BAF, bafilomycin; SCH, Sch-28080. *Significant difference from control (P < 0.05).

To investigate whether any other NHE isoform such as NHE2 contributes to the remaining component of JHCO3 in NHE3 knockout mice, we examined the effects of the Na+/H+ exchange inhibitor ethylisopropylamiloride (EIPA). This inhibitor was added to the lumen perfusate at a concentration of 100 µM. As shown in Table 2 and Fig. 1, EIPA significantly decreased JHCO3 and Jv by 40 and 47%, respectively, in wild-type mice. These results are similar to previous studies in rat proximal tubule (25, 35). In contrast, EIPA did not reduce either JHCO3 or Jv in NHE3 knockout mice. These results indicate that other NHE isoforms such as NHE2 do not play a role in mediating HCO-3 absorption in the absence of NHE3. It may be noted that EIPA did not reduce JHCO3 and Jv in the wild-type mice to the levels observed in the knockout mice. This can be explained by the known resistance of NHE3 to amiloride analogs in the presence of physiological Na+ concentrations (12, 18, 25, 35), resulting in incomplete inhibition of NHE3 activity in the wild-type mice.

To evaluate the contribution of the H+-ATPase to mediating HCO-3 absorption in the proximal tubules of wild-type and NHE3 null mice, we studied the effect of 1 µM bafilomycin A1 (17, 32, 34). The results in Table 2 and Fig. 1 show that bafilomycin decreased JHCO3 in wild-type mice by 22%, indicating that, under physiological conditions, a significant fraction of proximal tubule HCO-3 absorption is mediated by the H+-ATPase. This result is also consistent with previous findings that a similar fraction of HCO-3 absorption in the proximal tubule of the rat is mediated by a Na+-independent and/or bafilomycin-sensitive mechanism (11, 32).

Fractional inhibition of JHCO3 by bafilomycin was far greater (59%) in NHE3 null mice compared with wild-type controls, indicating that the major fraction of the JHCO3 that persists in the absence of NHE3 activity is mediated by the H+-ATPase. However, the absolute decrement in JHCO3 (25 vs. 30 pmol · min-1 · mm-1) was virtually the same in NHE3 knockout and wild-type animals, indicating a lack of significant compensatory upregulation of H+-ATPase in the NHE3 null mice. Interestingly, although there was a trend for bafilomycin to reduce Jv, this inhibition did not reach statistical significance in either wild-type or NHE3 null mice.

Finally, we studied the effect of the inhibitor Sch-28080 to assess the role of H+-K+-ATPase (14) in mediating proximal tubule HCO-3 and fluid absorption in wild-type and NHE3 null mice. The data in Table 2 and Fig. 1 demonstrate that 10 µM SCH-28080 failed to inhibit JHCO3 and Jv in either wild-type or NHE3 knockout mice. These findings indicate that H+-K+-ATPase activity does not contribute significantly to proximal tubule HCO-3 absorption in wild-type or NHE3 null mice.


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We have evaluated the relative contributions of apical H+ extrusion mechanisms to mediating HCO-3 reabsorption in the proximal tubules of NHE3 null mice. First, we confirmed the previous findings (27) that a large fraction (50-60%) of HCO-3 and fluid absorption in the microperfused mouse proximal tubule is dependent on the operation of the apical NHE isoform NHE3. These findings are also in accord with free-flow micropuncture data indicating marked impairment of fluid reabsorption in the proximal tubule of NHE3 null mice (20).

Second, we could detect no EIPA-sensitive component of JHCO3 or Jv in the proximal tubules of NHE3 null mice. Because NHE2 is more sensitive to amiloride analogs than is NHE3 (12), these findings indicate that NHE2 does not appreciably contribute to HCO-3 absorption, although its expression has been detected in the proximal tubule (39, 40).

Third, we found a significant component of HCO-3 absorption mediated by bafilomycin-sensitive H+-ATPase in the proximal tubules of both wild-type and NHE3 null mice, consistent with previous demonstrations of Na+-independent and/or bafilomycin-sensitive acidification in this nephron segment (11, 13, 32, 33). The contribution of apical membrane H+-ATPase to proximal tubule acidification is also supported by studies of H+ transport in isolated brush-border vesicles (17), the observation of Na+-independent acid extrusion from intact cells (19, 34), and the expression of H+-ATPase on the apical membrane as detected by immunostaining (8). However, we detected no upregulation of the bafilomycin-sensitive component of HCO-3 absorption in the proximal tubule of the NHE3 null mice.

Fourth, we were unable to detect a component of Sch-28080-sensitive HCO-3 absorption, arguing against a significant role for H+-K+-ATPase in mediating proximal tubule acidification in either wild-type or NHE3 null mice, although Sch-28080-sensitive K+-ATPase activity has been detected in this nephron segment (41). It should be noted that the concentration of Sch-28080 used in our experiments, 10 µM, should have been sufficient to inhibit at least 80% of the K+-ATPase activity that had been identified in the proximal tubule (41).

A component of proximal tubule HCO-3 reabsorption (21 pmol · min-1 · mm-1) persisted in the NHE3 null mice in the presence of bafilomycin (see Table 2 and Fig. 1), corresponding to 19% of the control rate of HCO-3 absorption in wild-type mice. There are several possible explanations for this small remaining component of inhibitor-insensitive HCO-3 absorption in the NHE3 null mice. First, a passive driving force for JHCO3 was present in these experiments because plasma HCO-3 concentration was reduced to ~20 mM in NHE3 null mice, whereas the concentration of HCO-3 in the tubule microperfusion solution was 25 mM. Assuming a mean transtubular HCO-3 gradient of 3 mM (Table 2), a negligible transtubular potential difference (15), and a HCO-3 permeability of 1.6 nl · mm-1 · min-1 as found in the rat proximal tubule (2), we calculate that only a minor fraction (5 pmol · min-1 · mm-1) of the remaining JHCO3 can be attributed to passive transport.

A second possible explanation for the remaining component of inhibitor-insensitive HCO-3 absorption in NHE3 null mice is the use of insufficient inhibitor concentrations to abolish H+-ATPase, H+-K+-ATPase, or non-NHE3-mediated Na+/H+ exchange. In the case of bafilomycin, a concentration of 15 nM is sufficient to abolish ATP-stimulated H+ transport in rat renal brush-border vesicles (17), suggesting that the concentration of 1 µM used in the present study should have been adequate to block H+-ATPase activity completely. However, it is possible that the inhibitor is absorbed in the perfused segment so that its concentration decreases, thereby resulting in incomplete inhibition of H+-ATPase activity. As mentioned above, 10 µM Sch-28080 should have been sufficient to reduce H+-K+-ATPase >80% in the proximal tubule (41), yet no inhibition by this agent was observed. NHE2, the only apical NHE isoform other than NHE3 so far identified, should have been significantly inhibited by 100 µM EIPA (12), but no EIPA inhibition of JHCO3 was observed in NHE3 null mice. Taken together, these considerations indicate that H+-K+-ATPase and non-NHE3-mediated Na+/H+ exchange are not likely to have contributed substantially to the inhibitor-insensitive JHCO3 in NHE3 null mice.

Third, it should be noted that acetate was present in the microperfusion solution used in these studies. Absorption of acetate via Na+-acetate cotransport in parallel with recycling of acetate back into the lumen by nonionic diffusion has been shown to effect net acid extrusion across the apical membrane of proximal tubule cells (22). However, acetate was found to inhibit rather than stimulate transtubular HCO-3 absorption (16), so that this mechanism is unlikely to account for significant HCO-3 absorption in NHE3 null mice. Alternatively, it is possible that secretion from blood to lumen of some other organic anion that crosses the apical membrane by nonionic diffusion or anion/OH- exchange may contribute to proximal acidification in these experiments (4).

Finally, despite a profound impairment of proximal tubule HCO-3 absorption capacity in NHE3 null mice, we confirmed previous findings (27) that only a mild metabolic acidosis is present in these animals. Although, as described above, we detected no upregulation of acidification mechanisms in the proximal tubule itself, at least two other compensatory responses to limit acidosis have so far been identified. First, there is a marked reduction in glomerular filtration rate in NHE3 null mice due to tubuloglomerular feedback (20). This in turn would be expected to reduce the filtered load of HCO-3 and thereby limit the delivery of HCO-3 out of the proximal tubule even in the presence of a reduced capacity for HCO-3 absorption. Second, HCO-3 absorption capacity is increased in cortical and outer medullary collecting ducts of NHE3 null mice due to upregulation of H+-K+-ATPase isoforms (21).


    ACKNOWLEDGEMENTS

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


    FOOTNOTES

Portions of the study were previously published in abstract form (37).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. S. Aronson, Dept. of Medicine/Nephrology, Yale School of Medicine, 333 Cedar St., PO Box 208029, New Haven, CT 06520-8029 (E-mail: peter.aronson{at}yale.edu).

Received 19 January 1999; accepted in final form 12 May 1999.


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Am J Physiol Renal Physiol 277(2):F298-F302
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