Novel Schering and ouabain-insensitive potassium-dependent proton secretion in the mouse cortical collecting duct

Snezana Petrovic1,3, Zachary Spicer2, Tracey Greeley1, Gary E Shull2, and Manoocher Soleimani1,3

Departments of 1 Medicine and 2 Molecular Genetics, Biochemistry, and Infectious Diseases, University of Cincinnati, and 3 Veterans Administration Medical Center, Cincinnati, Ohio 45267


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The intercalated (IC) cells of the cortical collecting duct (CCD) are important to acid-base homeostasis by secreting acid and reabsorbing bicarbonate. Acid secretion is mediated predominantly by apical membrane Schering (SCH-28080)-sensitive H+-K+- ATPase (HKA) and bafilomycin-sensitive H+-ATPase. The SCH-28080-sensitive HKA is believed to be the gastric HKA (HKAg). Here we examined apical membrane potassium-dependent proton secretion in IC cells of wild-type HKAg (+/+) and HKAg knockout (-/-) mice to determine relative contribution of HKAg to luminal proton secretion. The results demonstrated that HKAg (-/-) and wild-type mice had comparable rates of potassium-dependent proton secretion, with HKAg (-/-) mice having 100% of K+-dependent H+ secretion vs. wild-type mice. Potassium-dependent proton secretion was resistant to ouabain and SCH-28080 in HKAg knockout mice but was sensitive to SCH-28080 in wild-type animals. Northern hybridizations did not demonstrate any upregulation of colonic HKA in HKAg knockout mice. These data indicate the presence of a previously unrecognized K+-dependent SCH-28080 and ouabain-insensitive proton secretory mechanism in the cortical collecting tubule that may play an important role in acid-base homeostasis.

hydrogen-potassium-adenosinetriphosphatase; kidney; mouse knockout; acid-base homeostasis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVE PROTON SECRETION by the collecting duct (CD) is coupled, in part, to active K+ absorption via a membrane-bound adenosinetriphosphatase (H+-K+-ATPase) (24, 32). The H+-K+-ATPase (HKA) that is expressed in renal CD under normal conditions shows striking molecular, biochemical, and physiological similarities to the HKA found in gastric parietal cells, which is responsible for the secretion of acid into the gastric lumen (21, 24, 32). This exchanger has been referred to as gastric HKA (HKAg) and is sensitive to inhibition by SCH-28080 (24, 32). A distinct but structurally related HKA is expressed in the distal colon, which mediates active K+ reabsorption (6, 15). This transporter is called colonic HKA (HKAc) or nongastric HKA and is sensitive to inhibition by ouabain (24). Molecular studies indicate expression of both HKAg and HKAc in CD (24, 32). Functionally they are thought to be involved in H+ secretion and/or K+ reabsorption but appear to be regulated differentially (24, 32). Furthermore, HKAc may also mediate the exchange of extracellular K+ for intracellular Na+ or NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (5, 11), consistent with this transporter working as a cation/K+ exchange mode.

To better understand the role of HKAg in acid-base regulation, transgenic mice deficient in this gene were examined. HKAg null mice have severe achlorhydria (25), consistent with the role of this transporter as the major acid-secreting process in the parietal cells of the stomach. The HKAg-deficient mice did not show any significant abnormality in systemic acid-base balance or serum potassium (25), despite the fact that this transporter plays an important role in net bicarbonate reabsorption in the collecting ducts of mouse kidney (12, 13). These results suggest that another acid-secreting transporter(s) is likely upregulated in the kidneys of HKAg null mice. To explore this issue further, renal cortical collecting ducts (CCDs) of wild-type or HKAg knockout mice were isolated and perfused, and their alpha - and beta -intercalated cells (ICs) were examined. The results indicate that a novel exchanger, which is distinct from HKAc, is upregulated in HKAg null mice and maintains the K+-dependent H+ secretion at a comparable level to wild-type animals.


    METHODS
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Animals. Wild-type and HKAg knockout mice were used for these experiments. HKAg knockouts were described recently (25). Animals were allowed free access to food and water and were studied at 3-6 mo of age. For potassium depletion studies, animals were placed on a potassium-free diet (11) for 19 days.

Isolation of CCDs. Animals were killed by intraperitoneal injection of 50 mg/kg pentobarbital sodium. Kidneys were quickly removed and placed in ice-cold dissection medium (solution 1, Table 1). Thin (~1 mm) slices were obtained and transferred to the dissection chamber. The temperature of the dissection medium in the chamber was kept at 4°C. Bovine serum albumin, 0.1%, was added to the dissection medium to prevent sticking of dissected tubules to the glass and forceps. CCDs were obtained by free-hand dissection out of cortical medullary rays. Tubules were measured using an eyepiece micrometer and generally were 0.3-0.5 mm in length. CCDs were distinguished from nearby proximal straight tubules by their approximately one-third smaller diameter and more turbid appearance compared with the "ground-glass" appearance of the proximal straight tubules. Thick ascending limbs, on the other hand, are thinner than CCDs and have a fragile, homogeneous, and slightly shiny appearance in reflected light.

                              
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Table 1.   Solutions used for microperfusion experiments

In vitro microperfusion. Dissected tubules were quickly transferred to a 1.5-ml temperature-controlled specimen chamber mounted on an inverted Zeiss Axiovert S-100 microscope (Carl Zeiss, Thornwood, NY). Tubules were perfused using concentric glass pipettes according to the method of Burg and Green (1) with modifications (19, 27) at 4.5 cmH2O pressure. Solutions that were used to perfuse and bathe the tubules are listed in Table 1. All solutions were delivered to the specimen chamber in CO2- and O2-impermeable tubing (Cole Palmer, Chicago, IL) by a peristaltic pump (Peristar, WPI, Sarasota, FL) at a rate of 1 ml/min. The chamber had a lid to minimize evaporation and heat loss and maintain constant gas pressure and pH. Chamber pH was frequently checked on a model B 213 Horiba pH meter that allows measurement of pH in small samples.

To identify damaged cells, 0.07 mg/ml Fast Green dye (Sigma, St. Louis, MO) was added to the perfusate. Damaged cells are stained green by Fast Green dye. Tubules were carefully inspected and discarded if green-staining cells or a perfusate leak were found.

Intracellular pH measurements. After 20-min equilibration in solution 2, each tubule was perfused with 6 µM of the fluorescent pH indicator bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) for 15 min. When BCECF-AM is introduced from the luminal side of a CCD, intercalated, but not principal, cells take up the dye (29). Charged and fluorescent molecules of the dye are trapped in the cell after cleavage of the ester moiety by cytoplasmic esterases. Another 10-15 min were allowed for the dye washout. Fluorescent measurements were done on a Zeiss Axiovert S-100 inverted microscope equipped with the Attofluor RatioVision Digital Imaging System (Attofluor, Rockville, MD). Achroplan ×40/0.8 water objective with 3.6-mm working distance was used. Excitation wavelengths were alternated between 488 and 440 nm, and emission was measured at 520 nm. Background fluorescence was measured before the dye was introduced to the tubule and automatically subtracted from all subsequent measurements. The Attofluor Digital Imaging System allows for the control of light source intensity. Balance between the light source intensity and the camera gain optimizes fluorescent imaging while at the same time applies the smallest light intensity that is sufficient to excite the dye. This helps to minimize photobleaching and photodamage of the tubule cells, which can be substantial (29).

To avoid tubule movements and out-of-focus fluorescence, the free end of the tubule was allowed to loosely adhere to the poly-L-lysine-covered part of the chamber coverslip (28). Only cells in sharp focus in the tubule wall were examined. Images were taken in duplicates at 2-s intervals. Attofluor RatioVision software allowed for "regions of interest" to be applied to individual cells so that multiple cells in a single tubule were simultaneously examined. Generally, three to eight cells were examined per tubule. Only one tubule per animal was used. Digitized images were analyzed by using Attograph software. At the end of each experiment, intracellular calibration was performed by using the high-K+-nigericin method of Thomas et al. (26). Calibration solutions were varied from pH 6.5 to 7.8, and calibration points were fitted to a linear regression curve, which was then used for conversion of calculated ratios to cell pH.

Experimental procedures. After a stable baseline cell pH reading in bicarbonate-buffered solution (solution 2) was achieved, the bath solution was switched to a chloride-free solution (solution 3) to distinguish between the IC types. As shown previously (14, 18, 20, 30), removal of bath Cl- causes alkalinization of alpha -ICs due to blocking and/or reversing the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. At the same time, beta -ICs acidify, because this maneuver stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit via the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. Basolateral Cl- conductance (14, 18, 20, 30) of the beta -ICs allows for intracellular Cl- to leave the cell on basolateral Cl- removal. This adds to the apical membrane Cl- gradient and stimulates apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit. Both the alkalinization of alpha -ICs as well as the acidification of beta -ICs is reversed on addition of Cl- to the bath.

After identification of the IC type, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was removed from the bath and the lumen (solution 4). Also, K+ was removed from the lumen to block H+-K+-ATPase (solution 5). Luminal H+-ATPase was blocked by adding 170 nM bafilomycin A1 to the luminal solution. Each tubule was equilibrated in those solutions for 20 min and then acidified by addition of 15 mM NH4Cl (solution 6) to the bath. Basolateral Na+/H+ exchange was blocked with 40 µM ethylisopropylamiloride (EIPA) and basolateral H+-ATPase of beta -ICs with 170 nM bafilomycin A1. The tubules were bathed in a solution containing NH4Cl for 3 min. The bath solution was then switched back to a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free HEPES-buffered solution (solution 4) with 40 µM EIPA and 170 nM bafilomycin A1. The lumen was kept K+ free, and luminal H+-ATPase was blocked with 170 nM bafilomycin A1. Under these conditions, no cell pH recovery was observed. The luminal solution was then switched to solution 4 (containing 5 mM K+) and 170 nM bafilomycin A1. K+-dependent cell pH recovery was followed for the next 5-10 min. Inhibitors were then removed, and recovery without inhibitors was followed for the next 10 min as an index of cell viability. In a separate series of experiments designed to test the inhibitor profile of H+-K+-ATPase, 10 µM SCH-28080 or 1.5 mM ouabain was added to the appropriate luminal solution (solution 4).

RNA isolation and Northern hybridization. Total cellular RNA was extracted from whole kidney of wild-type or HKAg null mice (on normal or K+-free diet for 19 days) by the method of Chomczynski and Sacchi (2). In brief, 0.5-1 g of tissue was homogenized at room temperature in 10 ml Tri Reagent (Molecular Research Center, Cincinnati, OH). RNA was quantitated by spectrophotometry and stored at -80°C. RNA samples were fractionated on a 1.2% agarose-formaldeyde gel. The samples were transferred to a nylon membrane, cross-linked by ultraviolet light, and baked for 1 h. Hybridization was performed according to Church and Gilbert (3). The cDNA probes were labeled with 32P-deoxynucleotide using the RadPrime DNA labeling kit (Life Technologies). After hybridization, the membranes were washed, blotted dry, exposed to a PhosphorImager cassette at room temperature for 24-48 h, and read by PhosphorImager (Molecular Dynamics). The K+-free diet protocol was similar to previous studies reported from our laboratories (11). For HKAc, three PCR products from the rat alpha -subunit cDNA (nucleotides 135-515, 2369-2998, and 3098-3678; Ref. 6) were pooled and used as an isoform-specific probe.

Chemicals. All chemicals were obtained from Sigma unless specified otherwise. EIPA was dissolved in methanol as a 40 mM stock on the day of an experiment and diluted 1:1,000 for the final concentration of 40 µM. A stock solution of bafilomycin A1 was made fresh for each experiment in methanol, and 1:1,000 dilution was used to make the final concentration of 170 nM. Ouabain was directly added and dissolved in appropriate solutions at a final concentration of 1.5 mM. This concentration was used on the basis of studies indicating that HKAc activity was inhibited partially by 1 mM ouabain (24). Nigericin was dissolved in ethanol as a 10 mM stock and diluted 1:1,000 for the final concentration of 10 µM. BCECF-AM was obtained from Molecular Probes (Eugene, OR) and kept frozen in small aliquots of stock in DMSO. It was diluted 1:1,000 for the final concentration of 6 µM. SCH-28080 was kept frozen in small aliquots of 100 mM stock in methanol and was used with a dilution of 1:10,000 in the final concentration of 10 µM.

Statistics. Results are give as means ± SE. Statistical comparisons between the groups were performed according to Student's t-test for nonpaired data. The data were considered significant if P < 0.05.


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ABSTRACT
INTRODUCTION
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K+-dependent intracellular pH recovery in alpha -ICs and beta -ICs in wild-type mouse CCDs. The first set of experiments was designed to examine K+-dependent luminal proton secretion in each of the IC types (alpha  and beta ) in CCDs in wild-type animals. A representative tracing is shown in Fig. 1. At the beginning of each experiment, IC type was determined by basolateral Cl- removal in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The same cells were then followed throughout the experiment. Nine of 19 cells in 5 CCDs of wild-type mice alkalinized on basolateral Cl- removal [change in intracellular pH (Delta pHi) = 0.21 ± 0.01] and were therefore considered alpha -ICs. Ten of 19 cells in 5 CCDs acidified on basolateral Cl- removal (Delta pHi = -0.26 ± 0.02) and were considered beta -ICs.


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Fig. 1.   Representative tracings demonstrate the effect of 5 mM extracellular K+ on intracellular pH (pHi) recovery from an acute acid load in intercalated cells (ICs) in wild-type (WT) mouse. Upon identification of IC type (see METHODS and first part of tracing), HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was removed from lumen and bath for the rest of the experiment to render both luminal and basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters inactive. In the presence of bafilomycin A1 on both sides (an inhibitor of H+- ATPase) and ethylisopropylamiloride (EIPA) in the bath (an inhibitor of basolateral Na+/H+ exchange), removal of K+ from lumen blocked pHi recovery from an acid load. The return of 5 mM K+ to the lumen resulted in a significant pHi recovery from acidosis in both cell types. The rate of K+-dependent pHi recovery was similar in both cells: 0.069 pH units/min in alpha -ICs and 0.065 pH units/min in beta -ICs. Removal of all inhibitors allowed for faster recovery at the rate of 0.142 pH units/min in alpha -IC and 0.129 pH units/min in beta -ICs (to 7.27 and 7.28, respectively).

After the IC cell types were examined, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was removed from the luminal and basolateral sides. The Na+/H+ exchanger was blocked by addition of 40 µM EIPA to the bath solution. The H+-ATPase was blocked by addition of 170 nM bafilomycin A1 to both luminal and bath solutions, and K+ was removed from the lumen. Under these experimental conditions, NH4Cl prepulse acidified alpha -ICs from a baseline pHi of 7.35 ± 0.02 to a nadir pHi of 6.58 ± 0.03 (n = 9 cells in 5 CCDs) and beta -ICs from a baseline pHi of 7.33 ± 0.02 to a nadir pHi of 6.53 ± 0.03 (n = 10 cells in 5 CCDs). No pHi recovery was observed in either cell type in the absence of luminal K+. As demonstrated in Fig. 1 and summarized in Fig. 3, addition of 5 mM K+ to the lumen resulted in pHi recovery at a rate of 0.109 ± 0.01 pH units/min in alpha -ICs and 0.093 ± 0.01 pH units/min in beta -ICs (P > 0.05). These rates are similar to rates reported in previous studies in normal rat and rabbit ICs (22, 23, 31). Cells recovered to 6.98 ± 0.04 and 7.01 ± 0.03 in alpha -ICs and beta -ICs, respectively. Removal of the inhibitors (EIPA and bafilomycin from bath and bafilomycin from the perfusate) resulted in additional recovery to the baseline pH at a rate of 0.259 ± 0.01 and 0.268 ± 0.01 pH units/min in alpha -ICs and beta -ICs, respectively. The lack of pHi recovery to baseline levels after K+ addition is in agreement with observations by other investigators (22, 23, 31). One explanation that has been offered in this regard is that HKA activity might be regulated by intracellular H+ concentration (22).

Effect of SCH-28080 on K+-dependent pHi recovery in alpha -ICs and beta -ICs in wild-type mouse CCD. Using the same protocol as described above, we also examined the effect of HKAg inhibitor SCH-28080 on K+-dependent pHi recovery in alpha -ICs and beta -ICs of wild-type mouse CCD. A representative tracing is shown in Fig. 2, and the results are summarized in Fig. 3. In this group of experiments, 7 of 16 cells in 6 CCDs alkalinized on basolateral Cl- removal (Delta pHi = 0.25 ± 0.02) and were therefore considered alpha -ICs. Nine of 16 cells in 6 CCDs acidified on basolateral Cl- removal (Delta pHi = -0.24 ± 0.02) and were considered beta -ICs. In these experiments, the NH4Cl prepulse acidified alpha -ICs from a baseline pHi of 7.26 ± 0.035 to a nadir pHi of 6.64 ± 0.047 (n = 7 cells in 6 CCDs) and beta -ICs from a baseline pHi of 7.33 ± 0.027 to a nadir pHi of 6.49 ± 0.05 (n = 9 cells in 6 CCDs). In the absence of K+ in the lumen, no pHi recovery was observed in either cell type. As demonstrated in Figs. 2 and 3, the presence of 10 µM SCH-28080 in the lumen blocked the K+-dependent pHi recovery by >80% in both IC cell types. Rates of K+-dependent pHi recovery were 0.019 ± 0.005 and 0.012 ± 0.004 pH units/min in alpha -ICs and beta -ICs, respectively (P < 0.001, compared with the rates of recovery in the absence of SCH-28080). The HKAg is highly sensitive to SCH-28080, with the ATPase activity inhibited by >90% in the presence of 1 µM SCH-28080 (reviewed in Ref. 24). It has been shown that at concentrations >10 µM, SCH-28080 can inhibit other transporters such as Na+-independent H+-ATPase (16). Furthermore, recent data demonstrated that prolonged exposure of kidney CD cells to SCH-28080 as well as high concentrations of SCH-28080 cause ATP depletion (4), which in turn can have nonspecific inhibition of HKA activity. To avoid these complications, we did not try higher concentrations of SCH-28080. On removal of SCH-28080 and other inhibitors, all cells recovered to their baseline pHi at the rate of 0.275 ± 0.02 pH units/min in alpha -ICs and 0.253 ± 0.022 pH units/min in beta -ICs. In summary, the results in Figs. 1-3 demonstrate comparable K+-dependent, SCH-28080-sensitive H+-secreting activity in the apical membrane of both alpha -ICs and beta -ICs and are consistent with the activity of luminal HKAg in both cell types.


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Fig. 2.   Representative tracings demonstrating the effect of Schering (SCH)-28080 on K+-dependent pHi recovery in ICs of WT mouse. The identification of IC type is demonstrated in the first part of the tracings. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was removed from lumen and bath for the rest of the experiment to render HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent transporters inactive. In the presence of bafilomycin A1 on both sides and EIPA in the bath, removal of K+ from lumen blocked pHi recovery from an acid load. SCH-28080 blocked the K+-dependent pHi recovery in IC cells. Removal of the inhibitors allowed for fast recovery at the rate of 0.211 pH units/min in beta -IC and 0.223 pH units/min in alpha -IC.



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Fig. 3.   SCH-28080 inhibition of K+-dependent pHi recovery from acid load of ICs in WT mice CCD (summary of results). The effect of luminal K+ and SCH-28080 on the rate of pHi recovery in alpha - or beta -IC in wild-type mice is shown.

K+-dependent pHi recovery in alpha -ICs and beta -ICs in HKAg knockout mouse CCD. In the next series of experiments, we examined K+-dependent pHi recovery in both IC cell types in HKAg knockout mice CCDs. On the basis of their pHi response to basolateral Cl- removal, 12 of 25 cells (in 6 CCDs) were identified as alpha -ICs, and the remaining 13 were identified as beta -ICs. In the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, baseline pHi was comparable in alpha -ICs of HKAg knockout (7.353 ± 0.004, n = 12) and wild-type animals (7.346 ± 0.002, n = 9) (P > 0.05). Interestingly, basal pHi in beta -ICs was lower in HKAg knockout animals (7.265 ± 0.005, n = 13) compared with wild-type animals (7.326 ± 0.003, n = 10) (P < 0.05). Absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> did not significantly alter the baseline pHi levels (data not shown).

A NH4Cl prepulse acidified alpha -ICs from a baseline pHi of 7.26 ± 0.035 to a nadir pHi of 6.64 ± 0.047 (n = 12 cells in 6 CCDs). beta -ICs acidified from a baseline pHi of 7.33 ± 0.027 to a nadir pHi of 6.49 ± 0.05 (n = 13 cells in 6 CCDs). Similar to wild-type animals, in the absence of luminal K+, no pHi recovery was observed in either cell type. Surprisingly, as shown in representative tracings in Fig. 4 and summarized and compared with wild-type mice in Fig. 5, both IC cell types in the CCDs of HKAg knockout mice demonstrate luminal K+-dependent H+-secreting activity comparable to that of wild-type mice: 0.118 ± 0.01 pH units/min (n = 12) in alpha -ICs and 0.102 ± 0.01 pH units/min in beta -ICs (n = 13) (P > 0.05 compared with alpha -ICs and beta -ICs without SCH-28080, respectively). On addition of K+ to the lumen, cells recovered to pHi 7.05 ± 0.02 in alpha -ICs and 7.06 ± 0.03 in beta -ICs. Removal of the inhibitors resulted in further pHi recovery to the baseline pH at the rate of 0.228 ± 0.01 pH units/min in alpha -ICs and 0.249 ± 0.02 pH units/min beta -ICs.


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Fig. 4.   Representative tracings demonstrating the effect of 5 mM K+ on pHi recovery from acid load in ICs of gastric H+-K+-ATPase (HKAg) null mice. After the identification of alpha - and beta -cells (as demonstrated in the first part of the tracing), HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was removed from the experiments to render HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters inactive. In the presence of bafilomycin A1 on both sides and EIPA in the bath, removal of K+ from lumen prevented pHi recovery from acidosis. The return of 5 mM K+ to the lumen resulted in significant pHi recovery (to ~6.88) in both cells. Rate of K+-dependent recovery was 0.083 pH units/min in alpha -IC and 0.066 pH units/min in beta -ICs. Removal of the inhibitors allowed for additional recovery at the rate of 0.142 pH units/min in alpha -IC and 0.143 pH units/min in beta -IC.



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Fig. 5.   Rate of K+-dependent pHi recovery from acute acid load in ICs of WT and HKAg knockout (KO) mice (summary of results). The effect of luminal K+ on the rate of pHi recovery in alpha - or beta -IC of WT and KO mice is shown.

Effect of SCH-28080 on K+-dependent pHi recovery in alpha -ICs and beta -ICs in HKAg knockout mouse CCD. To characterize the K+-dependent H+ secretion in the CCDs of HKAg knockout mice, we sought to examine its sensitivity to SCH-28080 or ouabain. Data from the experiments examining the effect of SCH-28080 are shown as a representative tracing in Fig. 6 and are summarized in Fig. 7. Based on their pHi response to bath Cl- removal, 11 of 22 cells in 5 CCDs were identified as alpha -ICs, and 11 cells were considered beta -ICs. The NH4Cl prepulse acidified alpha -ICs from a baseline pHi of 7.29 ± 0.027 to a nadir pHi of 6.64 ± 0.019 (n = 11 cells). beta -ICs acidified from a baseline pHi of 7.27 ± 0.032 to a nadir pHi of 6.67 ± 0.027 (n = 11 cells). In the absence of K+ in luminal perfusate, no pHi recovery was observed in either cell type. However, and contrary to wild-type animals, SCH-28080 did not block the pHi recovery in the presence of 5 mM potassium in the lumen. This phenomenon was observed in both IC cell types. The rate of K+-dependent H+ secretion in the lumen of HKAg knockout mice CCDs was 0.097 ± 0.005 pH units/min in alpha -ICs (n = 11) and 0.094 ± 0.007 pH units/min in beta -ICs (11 cells in 5 CCDs). Compared with the cells in tubules of knockout mice perfused in the absence of SCH-28080, no significant difference was noted (P > 0.05 with or without SCH-28080), indicating that SCH-28080 did not affect the K+-dependent pHi recovery in ICs of knockout mice CCDs. pHi recovered to 6.99 ± 0.03 and 7.05 ± 0.03 in alpha -ICs and beta -ICs, respectively. Removal of the inhibitors resulted in additional recovery to the baseline pH at the rate of 0.251 ± 0.012 pH units/min in alpha -ICs and 0.243 ± 0.006 pH units/min in beta -ICs.


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Fig. 6.   Representative tracings demonstrating the effect of SCH-28080 on K+-dependent pHi recovery from acid load in ICs from a HKAg KO mice. H+-ATPase was blocked with bafilomycin A1 on both sides, and basolateral Na+/H+ exchange was blocked with EIPA. Interestingly, the presence of SCH-28080 did not prevent the K+-dependent pHi recovery. The cell recovered to pHi ~6.8. Rate of K+-dependent recovery was 0.078 pH units/min. Removal of the inhibitors allowed for additional recovery at the rate of 0.301 pH units/min to pHi 7.34.



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Fig. 7.   SCH-28080 does not inhibit K+-dependent pHi recovery from acid load in ICs of HKAg KO mice (summary of results). The effect of luminal K+ and SCH-28080 on the rate of pHi recovery in alpha - or beta -IC of HKAg KO mice is shown.

Effect of ouabain on K+-dependent pHi recovery in alpha -ICs and beta -ICs in HKAg null mouse CCD. The purpose of the next series of experiments was to examine the effect of ouabain on K+-dependent pHi recovery in CCDs of HKAg knockout mice. Figure 8 is a representative tracing, and Fig. 9 summarizes the results. In this group of experiments, 4 of 11 cells (from 2 CCDs) were identified as alpha -ICs, and 7 of 11 cells were considered beta -ICs. The NH4Cl prepulse acidified alpha -ICs from a baseline pHi of 7.33 ± 0.031 to a nadir pHi of 6.79 ± 0.005 (n = 4). beta -ICs acidified from a baseline pHi of 7.22 ± 0.03 to a nadir pHi of 6.71 ± 0.03 (n = 7). Similar to previous experiments, in the absence of K+ in the lumen, no pHi recovery was observed in either cell type. In the presence of 1.5 mM ouabain in the lumen, the rate of K+-dependent pHi recovery in IC cells of HKAg knockout mice remained unchanged vs. no ouabain. As indicated in Fig. 9, the pHi recovery rate in the presence of ouabain was 0.099 ± 0.007 pH units/min in alpha -ICs (n = 4) and 0.097 ± 0.005 pH units/min in beta -ICs (n = 7) of the HKAg knockouts. These were not statistically different vs. no ouabain in alpha -ICs and beta -ICs, respectively (P > 0.05) (Fig. 9). Cells recovered to 7.06 ± 0.02 in alpha -ICs and 6.99 ± 0.03 in beta -ICs. Removal of inhibitors resulted in additional recovery to baseline pHi levels at the rate of 0.236 ± 0.013 pH units/min in alpha -ICs and 0.234 ± 0.01 pH units/min in beta -ICs.


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Fig. 8.   Representative tracings demonstrate the effect of ouabain on K+-dependent pHi recovery in ICs from HKAg KO mice. H+-ATPase was blocked with bafilomycin A1 on both sides, and basolateral Na+/H+ exchange was blocked with EIPA. Under these conditions, K+ removal from lumen prevented pHi recovery from cell acidosis. Presence of ouabain at 1.5 mM did not inhibit the K+-dependent pHi recovery from acidosis. pHi recovered to ~7.2 in the presence of ouabain. Rate of K+-dependent recovery was 0.13 ph units/min in alpha -IC and 0.105 pH units/min in beta -ICs. Removal of the inhibitors allowed for additional recovery at a rate of 0.178 pH units/min in alpha -IC and 0.16 pH units/min in beta -ICs to pHi of 7.34 and 7.35, respectively.



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Fig. 9.   Ouabain does not inhibit K+-dependent pHi recovery from acid load in ICs of HKAg KO mice (summary of results). The effect of ouabain and luminal K+ on the rate of pHi recovery in HKAg KO mouse is shown.

RT-PCR and Northern hybridization studies in wild-type and HKAg null mouse. The gene-targeting strategy eliminated sequences encoding the catalytic phosphorylation site, which is essential for enzyme activity. In the stomach, the wild-type HKAg mRNA was eliminated (25), although there were trace levels of an ~1-kb mRNA encoding some of the NH2-terminal sequences. To test for the presence of HKAg mRNAs in kidneys of wild-type and mutant mice, RT-PCR analysis was performed using primers from exons 6 and 10. As shown in Fig. 10, a PCR product of the appropriate size (653 bp) was identified in wild-type kidney, but not in knockout kidney. These results confirm that these critical sequences, which span the major catalytic domain and the region required for apical sorting (10) of the pump, are absent in RNA from knockout kidney.


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Fig. 10.   RT-PCR analysis of RNA for the presence of HKAg. RT-PCR analysis was performed on total RNA isolated from kidneys of WT and HKAg KO animals using primers from exons 6 and 10. PCR products were size fractionated on 1.2% agarose gel and visualized by ethidium bromide staining. The results demonstrate a PCR product of expected size (653 bp) for mouse HKAg in the kidneys of WT but not HKAg KO animals. The specificity of the reaction is demonstrated by the absence of an RT product in the absence of reverse transcriptase (RT-). Bottom panel shows constitutive control beta -actin.

It is possible that in mice deficient in HKAg, the HKAc becomes upregulated and compensates for HKAg deficiency, as both transporters are expressed in the CD and mediate the exchange of luminal K+ for intracellular H+. Although the lack of effect of ouabain on K+-dependent H+ extrusion argues against such a possibility, a definite answer should come from expression studies. Accordingly, we examined the renal expression of HKAc by Northern hybridization. As indicated in Fig. 11, mRNA levels for HKAc remained undetectable in whole kidney of both HKAg knockout and wild-type animals on a normal diet (control groups). To determine the sensitivity of the HKAc assay, both HKAg knockout and wild-type animals were fed a K+-free diet according to established protocols (11) and examined after 19 days. As indicated in Fig. 11, both wild-type and HKAg null mice were able to upregulate their renal HKAc expression in response to K+ depletion. These results indicate that although the HKAg null mice can upregulate their HKAc in response to pathophysiological states, they do not upregulate it in response to HKAg gene deletion. Taken together, these results support the conclusion that a yet unrecognized, luminal, K+-dependent, SCH-28080- and ouabain-insensitive H+-secreting activity contributes to acid secretion in the CCD of HKAg null mice.


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Fig. 11.   Northern hybridizations of colonic HKA (HKAc) in whole kidney of WT and HKAg KO mice. As indicated, HKAc mRNA levels remained undetectable in WT and HKAg KO animals on a normal diet (control groups). Both WT and HKAg KO mice upregulated their HKAc mRNA in potassium depletion. The 28S rRNA is shown in the bottom of the blot as an index of RNA loading. RNA (30 µg/lane) was loaded in each lane.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies examine the K+-dependent luminal acid extrusion in CCDs of wild-type and HKAg null mice. HKAg null mice demonstrate an H+/K+ exchange activity that is comparable to wild-type animals (Figs. 1-5). Whereas SCH-28080 inhibits H+/K+ exchange activity in wild-type animals, H+/K+ exchange activity in the CCDs of null mice is resistant to inhibition by SCH-28080 (Figs. 6 and 7). H+/K+ exchange activity in null mice was also insensitive to inhibition by ouabain, a known inhibitor of HKAc (Figs. 8 and 9). Northern hybridizations did not demonstrate any upregulation of HKAc in HKAg null mice (Fig. 11).

HKAg is expressed in the CDs of mammalian kidneys, as supported by molecular, biochemical, and functional studies (24, 32). Studies on the inhibitor profile of HKAg demonstrate that this transporter is inhibited by SCH-28080 but not ouabain (24). Microperfusion studies in rabbit and rat kidney indicate that a significant portion of K+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in CCD is mediated via the SCH-28080-sensitive HKAg (24, 32). Studies in microperfused mouse CCD demonstrate that ~50% of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption is mediated via a K+-dependent process that is inhibited by SCH-28080 (12, 13). Taken together, these studies indicate that HKAg is responsible for a significant portion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the mouse kidney CD.

The present studies demonstrate that alpha -IC and beta -IC in mouse CCDs express apically oriented K+-dependent, SCH-28080-sensitive acid-secreting activity, strongly suggestive of functional HKAg. This is in agreement with functional studies in split-open rat and rabbit CCDs and microperfused rabbit CCDs (22, 23, 31). Interestingly, baseline pHi in beta -ICs of HKAg knockout mice was significantly lower compared with wild-type animals, whereas it was comparable in alpha -IC cells (see RESULTS). On the basis of present data, we cannot be sure about what causes this difference in beta -ICs or why there is not a difference in the basal pHi in alpha -ICs. We can only speculate that the lower basal pHi in beta -ICs in HKAg knockout mice raises the possibility that these cells are either pumping HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> out at higher rates or a H+-translocating transporter gets downregulated. Although both alpha - and beta -ICs seem to have apically oriented HKAg, it may serve different functions in these two cell types, or the way two cell types compensate for the lack of HKAg may be different.

HKAg null mice did not display any significant abnormality in systemic acid-base balance or serum K+ under baseline conditions (25). This suggests that acid-base transporter(s) distinct from HKAg is upregulated in the CDs of HKAg null mice. Comparable levels of K+-dependent pHi recovery in the CCDs of both animal groups are consistent with this hypothesis. Interestingly, the K+-dependent H+ secretion in the CCDs of HKAg null mice is not inhibited by classical HKA inhibitors such as SCH-28080 or ouabain, indicating the existence of a novel, yet unrecognized isoform of HKA in the kidney.

With respect to nongastric HKAs, HKAc is the well-known SCH-28080-insensitive, ouabain-sensitive isoform (7, 32). HKAc expression did not increase in HKAg null mice, indicating that this transporter is not responsible for the upregulation of HKA activity in this animal. Furthermore, the ouabain insensitivity of HKA activity in HKAg null mice points to an isoform distinct from HKAc. Recent functional studies in the colon have described the presence of another HKA isoform, which is insensitive to ouabain (9). No distinct HKA molecule that is insensitive to both SCH-28080 and ouabain has been identified.

Studies on HKAs in the kidney point to the discrepancies in the inhibitor profile of "nongastric" HKAs in heterologous expression systems and native tissues and have concluded that the presence of a novel isoform may account for the discrepancies (7, 24). Studies in cultured kidney cells (4) have questioned some of the inhibitor profile studies by demonstrating nonspecific effects of high concentrations of SCH-28080 on ATPase activity. To avoid any nonspecific effect of SCH-28080 on HKA transporters, the exposure time of perfused tubules to SCH-28080 in the present experiments was kept short.

Recently, Laroche-Joubert et al. (8) examined the properties of three functional K+-ATPases isoforms in microdissected rat nephron segments. They concluded that type II and III K+-dependent ATPase activities exhibit different sensitivity profiles to SCH-28080 and ouabain compared with the type I renal K+-ATPase. Type I K+-dependent ATPase is sensitive to SCH-28080 but is insensitive to ouabain and resembles HKAg. Type III is sensitive to ouabain but is insensitive to SCH-28080. They further found that pharmacological properties and tubular localization of type III K+-ATPase are not compatible with that of HKAc. They suggested that a new kidney HKA isoform might exist that may fit the properties of type III K+-ATPase. It is worth mentioning that an HKA that is insensitive to both SCH-28080 and ouabain has not been described in kidney epithelial cells. As such, the molecule mediating the H+/K+ exchange in apical membrane of ICs in HKAg knockout mice is distinct from all HKA isoforms described so far.

In conclusion, our results suggest that a novel acid-base transporter, distinct from HKAc, is upregulated in HKAg null mice and maintains the K+-dependent proton secretion at a comparable level to wild-type animals. This may account for the lack of any acid-base abnormality in HKAg null mice.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52821 (to M. Soleimani) and DK-50594 (to G. E. Shull), a merit review grant, and grants from Dialysis Clinic, Incorporated (to M. Soleimani).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Soleimani, Div. of Nephrology and Hypertension, Dept. of Internal Medicine, Univ. of Cincinnati, 231 Albert Sabin Way, MSB 5502, Cincinnati, OH 45267-0585 (E-mail: manoocher.soleimani{at}uc.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.

First published August 8, 2001; 10.1152/ajprenal.00124.2001

Received 18 April 2001; accepted in final form 2 August 2001.


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DISCUSSION
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