HCOminus 3 reabsorption in renal collecting duct of NHE-3-deficient mouse: a compensatory response

Suguru Nakamura1, Hassane Amlal1, Patrick J. Schultheis2, John H. Galla1, Gary E. Shull2, and Manoocher Soleimani1

Departments of 1 Internal Medicine and 2 Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Mice with a targeted disruption of Na+/H+ exchanger NHE-3 gene show significant reduction in HCO-3 reabsorption in proximal tubule, consistent with the absence of NHE-3. Serum HCO-3, however, is only mildly decreased (P. Schulties, 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), indicating possible adaptive upregulation of HCO-3-absorbing transporters in collecting duct of NHE-3-deficient (NHE-3 -/-) mice. Cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) were perfused, and total CO2 (net HCO-3 flux, JtCO2) was measured in the presence of 10 µM Schering 28080 (SCH, inhibitor of gastric H+-K+-ATPase) or 50 µM diethylestilbestrol (DES, inhibitor of H+-ATPase) in both mutant and wild-type (WT) animals. In CCD, JtCO2 increased in NHE-3 mutant mice (3.42 ± 0.28 in WT to 5.71 ± 0.39 pmol · min-1 · mm tubule-1 in mutants, P < 0.001). The SCH-sensitive net HCO-3 flux remained unchanged, whereas the DES-sensitive HCO-3 flux increased in the CCD of NHE-3 mutant animals. In OMCD, JtCO2 increased in NHE-3 mutant mice (8.8 ± 0.7 in WT to 14.2 ± 0.6 pmol · min-1 · mm tubule-1 in mutants, P < 0.001). Both the SCH-sensitive and the DES-sensitive HCO-3 fluxes increased in the OMCD of NHE-3 mutant animals. Northern hybridizations demonstrated enhanced expression of the basolateral Cl-/HCO-3 exchanger (AE-1) mRNA in the cortex. The gastric H+-K+-ATPase mRNA showed upregulation in the medulla but not the cortex of NHE-3 mutant mice. Our results indicate that HCO-3 reabsorption is enhanced in CCD and OMCD of NHE-3-deficient mice. In CCD, H+-ATPase, and in the OMCD, both H+-ATPase and gastric H+-K+-ATPase contribute to the enhanced compensatory HCO-3 reabsorption in NHE-3-deficient animals.

acid-base; proton-potassium-adenosinetriphosphatase; AE-1; proton-adenosinetriphosphatase; bicarbonate reabsorption; cortical collecting duct; outer medullary collecting duct; NHE-3 knockout


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MORE THAN 85% OF FILTERED HCO-3 is reabsorbed in the proximal tubule by a process of active acid secretion, with the remaining filtered HCO-3 being reabsorbed in the thick ascending limb and collecting duct (3, 13, 14, 15, 16). The acid-secreting transporters that are responsible for the reabsorption of HCO-3 in the proximal tubule are the Na+/H+ exchanger NHE-3 and the H+-ATPase, with NHE-3 mediating ~60% of total HCO-3 reabsorption in this nephron segment (3, 6, 14, 17, 29). The collecting duct reabsorbs 5-10% of total filtered HCO-3 and plays a major role in the final control of urine pH (15, 16, 18, 23). HCO-3 reabsorption in the cortical collecting duct (CCD) is predominantly mediated via the H+-ATPase (23, 25, 32), whereas in outer medullary collecting duct (OMCD), the gastric H+-K+-ATPase (HKAg) is the major transporter involved in HCO-3 reabsorption (21, 28, 33). A nongastric HKA (colonic HKA, or HKAc) is also expressed in the collecting duct but does not play a significant role in HCO-3 reabsorption under normal conditions (1, 10, 19, 21).

Recently, an NHE-3-deficient mouse was developed that shows a significant reduction in HCO-3 reabsorption in the proximal tubule (22). Serum HCO-3 concentration, however, was only mildly decreased (22), indicating possible adaptive upregulation of HCO-3absorbing transporters in collecting duct of NHE-3-deficient (NHE-3 -/-) mice. The purpose of the current experiments was to examine HCO-3-absorbing transporters in the CCD and OMCD of NHE-3-deficient mice.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HCO-3 transport measurement in CCD and OMCD. After removal, both kidneys of wild-type (WT) or NHE-3-deficient mice were decapsulated, sectioned into three to four cross sections per kidney, and immediately placed in a Petri dish containing dissecting solution. Each section was stripped from the papillary tip to the cortex into smaller wedges and transferred into a second Petri dish containing dissecting solution maintained at 14°C under a dissecting microscope. Segments of CCD and OMCD were dissected as before (11, 12). Tubules were then transferred to a Lucite chamber containing bathing solution (Table 1) initially at room temperature. One end of the tubule was pulled into an outer holding pipette. Once secure, the inner perfusion pipette was advanced, and the tubule was opened with a slight positive pressure. The opposite end of the tubule was then pulled into an outer collecting pipette. The tubules were warmed to 37°C in a temperature-controlled chamber and bathed with solution replaced every 30 min. Perfusion rates were maintained at 1-2 nl/min. Collections were made at 20- to 30-min intervals in a precalibrated constant-bore collection pipette. Three collections were made per each tubule. The collected samples were placed in a Petri dish under mineral oil. The solutions used are shown in Table 1. HCO-3-containing solutions were bubbled with 5% CO2-95% O2 gas. Bath pH was nominally 7.4. The osmolarity of the solutions was adjusted to 290 mosM by addition of sucrose.

                              
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Table 1.   Composition of solution

Inhibitors. To assess the contribution of different pathways of proton secretion for net HCO-3 reabsorption (JtCO2), three inhibitors were added to the perfusate. To block H+-ATPase, 50 µM diethylestilbestrol (DES) or 10 nM bafilomycin was used. The inhibitory effects of bafilomycin and DES were not additive (please see Fig. 5 later), indicating that DES inhibits JtCO2 via the bafilomycin-sensitive H+-ATPase. To inhibit HKAg, 10 µM Schering 28080 (SCH) was used. The inhibitory effects of SCH and DES on JtCO2 in the mouse collecting duct are additive (20), indicating inhibition of HKAg and H+-ATPase, respectively. Two inhibitors were tested in each collection. Three collections were made in each tubule, one without and two with an inhibitor. In all perfusions, the sequence of control and inhibitor was varied.

Measurement of tCO2 flux. tCO2 in nanoliter samples from collectate and perfusate was measured by microfluorometry (Nanoflow; World Precision Instruments). The JtCO2 (in pmol · min-1 · mm tubule length-1) across the tubule epithelium was calculated as
<IT>J</IT><SUB>tCO2</SUB> = (C<SUB>0</SUB>V<SUB>0</SUB> − C<SUB>1</SUB>V<SUB>1</SUB>)/<IT>L</IT>
where C0 is the concentration of tCO2 in the perfusion fluid (in pmol/nl), C1 is the concentration of tCO2 in the collected fluid (in pmol/nl), V0 is the perfusion rate (nl/min), V1 is the collection rate (in nl/min) (note that in the absence of vasopressin, V0 = V1), and L is the length of the tubule (in mm).

RNA isolation. Total cellular RNA was extracted from kidney (whole kidney, cortex, or medulla) by the method of Chomczynski and Sacchi (8). In brief, 0.2 g of tissue was homogenized at room temperature in 10 ml Tri Reagent (Molecular Research Center, Cincinnati, OH). RNA was extracted by phenol/chloroform, precipitated by isopropanol, and quantitated by spectrophotometry. RNA was stored at -80°C until used.

Northern hybridization. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna NT nylon membranes (MSI). Membranes were then cross-linked by ultraviolet light and baked as described. Hybridization was performed according to Church and Gilbert (9). The cDNA probe was labeled with 32P-labeled deoxynucleotides using the RadPrime DNA labeling kit (GIBCO-BRL). The membranes were washed, blotted dry, and screened by a Phosphor Imager screen (Molecular Dynamics). For HKAg, HKAc, and H+-ATPase Northern blots, rat cDNA-specific probes were used as before. For AE-1, a 650-bp cDNA (Sac I-Bgl II fragment) from rat AE-1 cDNA was used as specific probe. The hybridizations were performed under high-stringency conditions to prevent any cross-hybridization with other isoforms.

Statistical analysis. The data are expressed as mean ± SE where appropriate. For statistical analysis of the differences in the levels of mRNA expression, Phosphor Imager readings from three separate experiments were obtained and analyzed. Statistical analysis was determined using ANOVA. P < 0.05 was considered statistically significant.

Materials. 32P was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma Chemical (St. Louis, MO). The GIBCO-BRL RadPrime DNA labeling kit was purchased from Life Technologies.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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JtCO2 in CCD of WT and NHE-3 knockout mice. In the first series of experiments, JtCO2 in CCD of WT and NHE-3 knockout mice was examined. As shown in Fig. 1, JtCO2 was enhanced in NHE-3 mutant animals, with JtCO2 increasing from 3.42 ± 0.28 to 5.71 ± 0.39 pmol · min-1 · mm tubule-1 (P < 0.001, n = 6 and 7 for WT and deficient mice, respectively).


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Fig. 1.   Net HCO-3 reabsorption (JtCO2) in cortical collecting duct (CCD) of wild-type (WT, +/+) and NHE-3 knockout (-/-) mice. JtCO2 was measured in CCD of WT and NHE-3-deficient mice, according to METHODS. As indicated, JtCO2 increased in NHE-3-deficient mice (* P < 0.001).

Effect of inhibitors on JtCO2 in CCD of WT and NHE-3 knockout mice. To examine the contribution of HKA and H+-ATPase, respective specific inhibitors of these transporters were used. For HKAg and H+-ATPase inhibition, 10 µM SCH and 50 µM DES was added to the perfusate, respectively.1 Figure 2, A and B, show the inhibitory effects of DES and SCH on JtCO2 in CCD of WT or NHE-3 knockout mice. These data permit the calculation of SCH-sensitive and DES-sensitive JtCO2, which are summarized in Fig. 2, C and D. Figure 2C shows that the SCH-sensitive JtCO2 remained unchanged in CCD of NHE-3-deficient mice. Figure 2D shows that the DES-sensitive JtCO2 increased in CCD of NHE-3-deficient mice.


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Fig. 2.   Effect of inhibitors on JtCO2 (in pmol · min-1 · mm-1) in CCD of WT and NHE-3 knockout mice. A and B: effects of diethylestilbestrol (DES) and Sch-28080 (SCH) on JtCO2 in CCD of WT (A) or NHE-3 knockout mice (B). In WT mice (A), JtCO2 (n = 6) was decreased by both DES (1.42 ± 0.48; P < 0.007) and SCH (1.82 ± 0.24; P < 0.002) compared with controls (CON, 3.42 ± 0.28). In mutant mice (B), JtCO2 (n = 7) was also decreased by both DES (2.27 ± 0.32; P < 0.001) and SCH (4.12 ± 0.34; P < 0.01). These data permit calculation of the SCH-sensitive components (WT 1.60 ± 0.21; mutant 1.58 ± 0.22), which did not differ (P = not significant) (C), and the DES-sensitive components, which were higher (P < 0.02) in mutant (3.44 ± 0.23) than in WT mice (2.0 ± 0.25) (D). * P < 0.05.

JtCO2 in OMCD of WT or NHE-3 knockout mice. The purpose of the next series of experiments was to examine JtCO2 in OMCD of WT or NHE-3 knockout mice. As shown in Fig. 3, JtCO2 was enhanced in OMCD of NHE-3 mutant animals, with JtCO2 increasing from 8.84 ± 0.68 to 14.20 ± 0.60 pmol · min-1 · mm tubule-1 (P < 0.001, n = 5 and 6 for WT and deficient mice, respectively).


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Fig. 3.   JtCO2 in outer medullary collecting duct (OMCD) of WT or NHE-3 knockout mice. JtCO2 was measured in OMCD of WT and NHE-3-deficient mice, according to METHODS. JtCO2 increased in NHE-3-deficient mice (* P < 0.001).

Effect of inhibitors on JtCO2 in OMCD of WT or NHE-3 knockout mice. To examine the contribution of HKA and H+-ATPase, 10 µM SCH or 50 µM DES was added to the perfusate, respectively. Figure 4, A and B, shows the inhibitory effects of DES and SCH on JtCO2 in OMCD of WT or NHE-3-deficient animals. Calculated SCH-sensitive and DES-sensitive JtCO2 values are depicted Fig. 4, C and D, which shows that both the SCH-sensitive and the DES-sensitive JtCO2 increased in OMCD of NHE-3-deficient mice.


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Fig. 4.   Effect of inhibitors on JtCO2 (in pmol · min-1 · mm-1) in OMCD of WT or NHE-3 knockout mice. A and B: effects of DES and SCH on JtCO2 in OMCD of WT (A) or NHE-3-deficient mice (B). In WT mice (A), JtCO2 (n = 5) was decreased by both DES (5.89 ± 0.58; P < 0.02) and SCH (4.8 ± 0.78; P < 0.01) compared with control (CON) perfusions (8.84 ± 0.68). In NHE-3-deficient mice (B), JtCO2 (n = 6) was also decreased by both DES (9.45 ± 0.49; P < 0.001) and SCH (5.34 ± 0.52; P < 0.0001). Similar to the CCD, the calculated DES-sensitive component was greater (P < 0.04) in mutant (4.71 ± 0.59) than in WT (2.95 ± 0.28, D). However, unlike the CCD, the SCH-sensitive component was greater (P < 0.0001) in mutant (8.81 ± 0.52) than in WT mice (4.04 ± 0.30, C). * P < 0.05.

DES inhibits JtCO2 via the bafilomycin-sensitive H+-ATPase. To determine the specificity of DES inhibition on H+-ATPase, the effect of DES and bafilomycin, a known specific inhibitor of the H+-ATPase, was compared. As shown in Fig. 5, the inhibitory effects of bafilomycin and DES were not additive, indicating that DES decreases JtCO2 via inhibition of H+-ATPase.


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Fig. 5.   DES inhibits JtCO2 (in pmol · min-1 · mm-1) via the bafilomycin (BAF)-sensitive H+-ATPase. JtCO2 (n = 4) in tubules perfused with BAF only (2.0 ± 0.08) did not differ (P = not significant) from that in those perfused with both BAF and DES (1.75 ± 0.10).* P < 0.05.

AE-1 mRNA expression in kidneys of WT and NHE-3-deficient mice. HCO-3 transport across the basolateral membrane of CCD cells is predominantly mediated via the Cl-/HCO-3 exchanger AE-1, which is exclusively expressed on the basolateral membranes of acid-secreting intercalated cells (2, 24, 30). As shown in Fig. 6, AE-1 mRNA expression is increased in the whole kidney of NHE-3 knockout animals. Zonal examinations showed enhancement of AE-1 mRNA levels in the cortex of NHE-3 knockout mice (Fig. 6), consistent with enhanced HCO-3 reabsorption in CCD. The expression of AE-1 in the medulla remained unchanged (Fig. 6). The AE-1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression ratio is shown in Fig. 6 (bottom).


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Fig. 6.   Northern hybridization of kidney AE-1 in WT or NHE-3 knockout mice. Representative Northern blots of AE-1 mRNA expression in whole kidney, cortex, and medulla: ratio of AE-1 mRNA to GAPDH mRNA. Results indicate that AE-1 mRNA expression is increased in whole kidney and cortex of NHE-3 knockout mice but remained unchanged in medulla (n = 3 for each group). NS, not significant.

HKAg mRNA expression in kidneys of WT and NHE-3-deficient mice. To correlate the activity of the SCH-sensitive HKA with its mRNA expression, HKAg mRNA levels were measured in whole kidney, cortex, and medulla. As shown in Fig. 7, HKAg mRNA is decreased mildly in the whole kidney of NHE-3 knockout mice; however, the difference was not statistically significant. Zonal analysis showed that HKAg mRNA expression is increased in the medulla but mildly decreased in the cortex (Fig. 7). The expression ratio of HKAg mRNA to GAPDH mRNA is shown in Fig. 7 (bottom).


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Fig. 7.   Northern hybridization of kidney gastric H+-K+-ATPase (HKAg) in WT or NHE-3 knockout mice. Representative Northern blots of HKAg mRNA expression in the whole kidney, cortex, and medulla: ratio of HKAg mRNA to GAPDH mRNA. HKAg mRNA expression is increased in the renal medulla but is mildly decreased in the cortex (n = 3 for each group).

H+-ATPase mRNA expression in kidneys of WT and NHE-3-deficient mice. The expression of mRNA encoding the 31-kDa subunit of the H+-ATPase was examined in the whole kidney, cortex, and medulla. As shown in Fig. 8, H+-ATPase mRNA abundance remained unchanged in whole kidney, cortex, and medulla of NHE-3 knockout mice. The H+-ATPase mRNA to GAPDH mRNA expression ratio is shown in Fig. 8 (bottom).


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Fig. 8.   Northern hybridization of kidney H+-ATPase in WT or NHE-3 knockout mice. Representative Northern blots of H+-ATPase mRNA in the whole kidney, cortex, and medulla: ratio of H+-ATPase mRNA to GAPDH mRNA. H+-ATPase mRNA expression remains unchanged in both cortex and medulla of NHE-3 knockout mice (n = 3 for each group, P > 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The current experiments demonstrate that HCO-3 reabsorption is enhanced in CCD and OMCD of NHE-3 mutant mice (Figs. 1 and 3). The increase in HCO-3 reabsorption in CCD is predominantly via H+-ATPase. In OMCD, increased HCO-3 reabsorption is mediated via both HKAg and H+-ATPase (Figs. 2 and 4). Northern hybridizations indicated enhanced expression of AE-1 in the cortex (Fig. 6) and HKAg in the medulla (Fig. 7).

The distal nephron, and particularly CCD and medullary collecting duct, plays an essential role in reabsorbing the HCO-3 and final acid-base composition of the urine (15, 16, 23). In CCD, HCO-3 reabsorption occurs predominantly by type A intercalated cells (A-type IC) (23, 25, 32). Two acid-secreting transporters, HKAg and H+-ATPase, are expressed at the apical membrane of the A-type IC (4, 7) and are responsible for the majority of HCO-3 reabsorption in this nephron segment. B-type IC are involved in HCO-3 secretion, which is predominantly mediated via the apical Cl-/HCO-3 exchanger (16, 23, 26, 27). In B-type IC, both HKAg and H+-ATPase are expressed on the basolateral membrane domain (4, 7) and are predominantly involved with intracellular HCO-3 generation, which would then be secreted into the lumen via the apical Cl-/HCO-3 exchanger (16, 23). Our results indicate enhanced AE-1 mRNA expression in the cortex of NHE-3-deficient mice (Fig. 5), which is consistent with the finding of increased HCO-3 reabsorption in the CCD. Lack of upregulation of AE-1 mRNA expression in medulla, despite enhanced HCO-3 reabsorption in OMCD, suggests that either the amount of exchanger is not rate limiting for overall bicarbonate reabsorption in OMCD or the majority of AE-1 is originating from segments other than the medullary collecting duct (such as descending and ascending limb of Henle). This is in contrast to the cortex, where AE-1 expression is limited only to CCD.

Our results demonstrated enhancement of H+-ATPase activity in CCD and OMCD of NHE-3-deficient mice. Lack of alteration in H+-ATPase mRNA expression indicates that enhancement of this transporter in NHE-3 mutant mice is likely a posttranscriptional event. This conclusion is consistent with previous studies demonstrating that adaptation of H+-ATPase in acid-base disorders is via alteration in the rate of insertion (exocytosis) or retrieval (endocytosis) of the transporter to or from the membrane rather than via alterations in the rate of transcription (23). Studies examining the localization of H+-ATPase protein (membrane bound vs. cytosolic) by immunocytochemistry should provide a more definite answer to this question.

The signal responsible for upregulation of H+-ATPase in the collecting duct of NHE-3-deficient mice remains speculative at this time. The primary renal defect in NHE-3 mutant mice is decreased reabsorption of HCO-3 in the proximal tubule, with subsequent increased delivery to the distal tubule. In addition to the kidney, NHE-3 mutant mice show decreased reabsorption of HCO-3 in the small intestine (22). As a result of the HCO-3 absorption defect in the kidney proximal tubule and small intestine, NHE-3 mutant mice show a mild metabolic acidosis (22, 31). One likely signal involved in the upregulation of H+-ATPase is increased delivery of HCO-3 to the distal nephron. According to this scheme, H+-ATPase is upregulated and enhances HCO-3 reabsorption in the collecting duct of NHE-3 knockout mice which in turn attenuates HCO-3 loss in the urine. Alternatively, it is possible that H+-ATPase upregulation in CCD (and OMCD) is due to the mild acidosis that is present in NHE-3 knockout mice (22, 31). In support of this latter hypothesis, several studies have shown that metabolic acidosis upregulates HCO-3 absorption in A-type IC of CCD via increased apical H+-ATPase and basolateral Cl-/HCO-3 exchange (AE-1) activities (23, 26, 27). Increased apical H+-ATPase activity under these circumstances was shown to be via enhanced insertion of this pump into the luminal membrane of A-type IC (5, 26, 27).

HKAg shows differential regulation at mRNA level in cortex and medulla in NHE-3 mutant mice. HKAg mRNA levels are increased in the medulla but mildly decreased in the cortex of NHE-3 knockout mice (Fig. 7). Upregulation of HKAg mRNA in the medulla correlates with its increased activity in OMCD as measured by SCH-sensitive JtCO2, and indicates transcriptional upregulation (or increased mRNA stability) of this transporter in NHE-3 mutant mice. Lack of enhancement of HKAg mRNA in the cortex of NHE-3 mutant animals remains suspect. Immunocytochemical studies in CCD have indicated that HKAg and H+-ATPase are colocalized on the apical membranes of A-type IC and on the basolateral membranes of B-type IC (4, 7). Studies in rabbits have shown that the CCD adapts to metabolic acidosis by downregulating HCO-3 secretion in B-type IC cells via decreased apical Cl-/HCO-3 exchange activity (26). Downregulation of apical Cl-/HCO-3 exchange activity results in alkaline intracellular pH in B-type IC cells, which in turn can suppress the basolateral HKAg. It is possible that the lack of upregulation of HKAg mRNA in the cortex (Fig. 6) may reflect the net effect of suppression of basolateral HKAg and upregulation of apical HKAg. However, comparable SCH-sensitive HKA activity in CCD of WT and mutant mice (Fig. 2) does not support this conclusion.

In summary, JtCO2 is increased in both CCD and OMCD of NHE-3 knockout mice. In CCD, JtCO2 is increased predominantly via upregulation of H+-ATPase activity, whereas in OMCD, JtCO2 is increased via upregulation of both HKAg and H+-ATPase activities. Compensatory HCO-3 reabsorption in the collecting duct reduces HCO-3 wasting in NHE-3 knockout mice and helps to maintain acid-base homeostasis.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789, DK-52821, and DK-54430 (to M. Soleimani) and DK-50594 (to G. E. Shull) and by grants from Dialysis Clinic Incorporated (to M. Soleimani and J. H. Galla).


    FOOTNOTES

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.

1 Colonic H+-K+-ATPase mRNA remained undetectable in NHE-3 knockout animals. Furthermore, 1 mM ouabain in the perfusate had no effect on net HCO-3 transport in wild-type or knockout animals. Taken together, these results indicate that colonic H+-K+-ATPase does not have an increased role in the NHE-3 knockout mice.

Address for reprint requests and other correspondence: M. Soleimani, Division of Nephrology and Hypertension, Dept. of Internal Medicine, Univ. of Cincinnati, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.edu).

Received 16 December 1998; accepted in final form 12 March 1999.


    REFERENCES
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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