Novel regulation of cell [Na+] in macula densa cells: apical Na+ recycling by H-K-ATPase

János Peti-Peterdi1, Zsuzsa Bebok2, Jean-Yves Lapointe3, and P. Darwin Bell1

1 Nephrology Research and Training Center, Division of Nephrology, and 2 Division of Hematology and Oncology, Department of Medicine and Physiology, and Gregory Flaming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 3 Membrane Transport Research Group, University of Montreal, Montreal, Quebec, Canada H3C 3J7


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Na-K-ATPase is the nearly ubiquitous enzyme that maintains low-Na+, high-K+ concentrations in cells by actively extruding Na+ in exchange for K+. The prevailing paradigm in polarized absorbing epithelial cells, including renal nephron segments and intestine, has been that Na-K-ATPase is restricted to the basolateral membrane domain, where it plays a prominent role in Na+ absorption. We have found, however, that macula densa (MD) cells lack functionally and immunologically detectable amounts of Na-K-ATPase protein. In fact, these cells appear to regulate their cytosolic [Na+] via another member of the P-type ATPase family, the colonic form of H-K-ATPase, which is located at the apical membrane in these cells. We now report that this constitutively expressed apical MD colonic H-K-ATPase can function as a Na(H)-K-ATPase and regulate cytosolic [Na+] in a novel manner. This apical Na+-recycling mechanism may be important as part of the sensor function of MD cells and represents a new paradigm in cell [Na+] regulation.

macula densa; sodium-potassium-5'-adenosinetriphosphatase; colonic hydrogen-potassium-5'-adenosinetriphosphatase; sodium-binding benzofuran isophthalate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OVER THE LAST FEW YEARS, DIFFERENT members of the Na-K/H-K-ATPase gene family have been identified and localized in various epithelia within the kidney (10, 12, 25). There is a high abundance of Na-K-ATPase along the basolateral membrane of various nephron segments (11), with the highest activity occurring in the outer medulla (thick ascending limb; TAL). Na-K-ATPase is involved in the development and maintenance of transmembrane Na-K electrochemical gradients that, in epithelial cells, are necessary for Na+ reabsorption and K+ secretion. In terms of the two other subgroups of this ATPase family, different isoforms of both the gastric H-K-ATPase and the colonic (nongastric) H-K-ATPase exist in the kidney (12, 25) and are located mainly in the distal nephron. Presently, it is thought that these ATPases play a role in K+ absorption and urinary acidification.

It has been thought that Na-K-ATPase activity and, consequently, the capacity for transepithelial NaCl transport are relatively low in macula densa (MD) cells, at least compared with the adjacent cortical TAL (cTAL). This was based, in part, on previous work (21) that demonstrated very low Na-K-ATPase activity in MD cells. This finding is intriguing, because these cells are located at the end of cTAL and act as sensor cells, detecting changes in luminal NaCl concentration ([NaCl]L) and sending signals to the mesangial-afferent arteriolar complex (tubuloglomerular feedback; TGF) (20). MD cell signaling has been shown to involve apical NaCl transport mechanisms, including a furosemide-sensitive Na+-2Cl--K+ cotransporter (3, 19). Also, the type 2 Na+/H+ exchanger isoform (NHE2), another Na+-entry pathway, is located at the apical membrane of MD and cTAL cells (15). It has been suggested that transport-induced secondary changes in intracellular [NaCl] are critically important in MD cell signaling. This controversy, i.e., importance but scarcity of NaCl transport, suggests a unique way of Na+ handling in MD cells. Therefore, the purpose of these studies was to measure transport-related changes in MD intracellular [Na+] ([Na+]i). For comparison, similar transport measurements were obtained in adjacent cTAL cells. These studies demonstrate that MD cells possess an apical H(Na)-K-ATPase that may be the primary Na+-efflux pathway for these cells. Similarity in pharmacological profiles of the MD and distal colon H-K-ATPases indicates that MD cells express a form of the colonic H-K-ATPase.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tubule and colonic crypt preparation. We isolated and perfused cTALs with attached glomeruli dissected from kidneys of rabbits kept on a standard diet [15% rabbit diet (W) 8630, Teklad, Madison, WI] as described earlier (14, 15). This preparation contains the otherwise inaccessible MD cells and allowed us to manipulate the composition of the tubular fluid at the apical side (perfusate) independently from the basolateral interstitium (bath). The dissection solution was a modified Ringer solution composed of (in mM) 25 NaCl, 5 KCl, 1 MgSO4, 1.6 Na2HPO4, 0.4 NaH2PO4, 1.5 CaCl2, 5 D-glucose, 5 HEPES, and 125 N-methyl-D-glucamine-cyclamate and adjusted to a pH of 7.4. Tubules were cannulated and perfused with this same Ringer solution. The preparation was bathed in 150 mM [NaCl] Ringer solution continuously aerated with 100% O2 and exchanged at a rate of 1 ml/min. Temperature was maintained at 37oC. Individual crypts of the distal colon from rabbit were also hand-dissected and perfused with methods similar to those used in perfusing kidney tubules.

[Na+]i measurement. [Na+]i of MD, cTAL cells, and the upper third of colonic crypt epithelial cells, known to express the H-K-ATPase (18), was measured with dual-excitation wavelength fluorescence microscopy (Photon Technologies, Princeton, NJ) by using the fluorescent probe sodium-binding benzofuran isophthalate (SBFI; Teflabs, Austin, TX) with techniques similar to those described for Ca2+ and intracellular pH (pHi) measurements (14, 15). SBFI fluorescence was measured at an emission wavelength of 510 nm in response to excitation wavelengths of 340 and 380 nm, alternated at a rate of 25 Hz by a computer-controlled chopper assembly. An adjustable photometer sampling window (15) was positioned over the whole MD plaque (consisting of ~15-20 cells), and emitted photons were detected by a Leitz photometer that was modified for photon counting. Magnification was ×400 by using an Olympus ×40 UVFL lens. Cells were loaded with the dye by adding SBFI-acetoxymethyl ester (AM; 20 µM) dissolved in DMSO to the luminal perfusate. The nonionic surfactant, Pluronic F-127 was added (1 mg/ml) to DMSO to facilitate loading, which required ~15 min, and then luminal SBFI-AM was removed. After a further ~15 min of exposure to the control perfusion solution, fluorescence intensities for both wavelengths stabilized at constant levels. SBFI fluorescence ratios (340/380 nm) were converted into [Na+]i values after permeabilizing of cell membranes on both sides of the tubule with 10 µM nigericin+monensin and equilibration of [Na+]i with ambient [Na+] in a stepwise manner between 0 and 150 mM. SBFI fluorescence was highly sensitive to the ambient [Na+] and was linear between 0 and 100 mM [Na+]i in both MD and cTAL cells.

Immunohistochemistry. Rabbit kidneys were perfusion-fixed, and tissue sections were processed as described earlier (15). Sections were blocked with goat serum (1:25) and incubated for 1 h with the highly specific monoclonal antibody alpha 6F (Developmental Studies Hybridoma Bank, University of Iowa; no dilution of the supernatant), which recognizes the Na-K-ATPase alpha 1-subunit in various species (2). This was followed by a 40-min incubation with fluorescein-conjugated goat anti-mouse IgG (Vector Labs, 1:200). Sections were mounted with Vectashield media containing 4,6-diamino-2-phenylindole (DAPI) for nuclear staining (Vector Labs). Tissue sections were examined with an Olympus IX70 inverted epifluorescence microscope using a UApo/340 ×40 objective. Images were captured using a SenSys digital camera and IPLab Spectrum software equipped with a power microtome (Signal Analytics).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transport-related dynamics of [Na+]i in MD and cTAL cells. Resting [Na+]i in MD cells, in the presence of 25 mM [NaCl]L and 150 mM NaCl in the bath, was 29.8 ± 4.4 mM (n = 12). As demonstrated in a representative recording (Fig. 1A) and summarized in Fig. 1B, increasing [NaCl]L from 25 to 150 mM caused a substantial increase in MD [Na+]i, that plateaued at an [Na+]i of ~70 mM. In contrast, baseline [Na+]i in adjacent cTAL cells was 12.7 ± 1.6 mM (n = 16) and increased by only 23.0 ± 6.9 mM in response to 150 mM [NaCl]L. As depicted in Fig. 1B, addition of furosemide, a blocker of Na+-2Cl--K+ cotransport, to the lumen significantly reduced resting [Na+]i and attenuated the [NaCl]L-induced elevations in [Na+]i in both cell types. This finding supports the notion that the primary mechanism for apical entry of NaCl in MD and cTAL cells is the Na+-2Cl--K+ cotransporter. In addition, as shown in Fig. 2, there is a sigmoidal relationship between [Na+]i and [NaCl]L, which plateaued at 60 mM [NaCl]L. This is consistent with the known affinity of the cotransporter for [Cl]L and is also consistent with the relationship between [NaCl]L and TGF signaling. It should also be mentioned, however, that 150 mM [NaCl]L, in the presence of luminal furosemide, slightly but significantly increased [Na+]i in both MD and cTAL cells, most probably via the apical Na+/H+ exchanger NHE2. Consistent with this suggestion is that the Na+/H+ exchanger blocker HOE-642, like furosemide, reduced both baseline [Na+]i and the magnitude of 150 mM [NaCl]L-induced elevations in MD [Na+]i (Fig. 3). However, HOE-642 was less effective compared with furosemide.


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Fig. 1.   Luminal NaCl concentration ([NaCl]L)-dependent changes in intracellular Na+ concentration ([Na+]i) in macula densa (MD) and cortical thick ascending limb (cTAL) cells. A: representative recordings demonstrate a rapid, sustained, and reversible increase in [Na+]i in both cell types when [NaCl]L was increased from 25 to 150 mM. B: effect of the diuretic furosemide (50 µM) on [Na+]i. , Control; , furosemide. Values are means ± SE; n = 6 each. L, luminal. *,#: P < 0.05, 25 and 150 mM [NaCl]L, respectively, compared with control values (t-test for 2 samples).



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Fig. 2.   [NaCl]L-dependent elevations in MD [Na+]i. Values are means ± SE; n = 5.



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Fig. 3.   Examples of recordings from MD preparations during [NaCl]L-dependent elevations in MD [Na+]i in the presence of the transport blockers furosemide (50 µM), HOE-642 (100 µM), and ouabain (1 mM) in the perfusate.

Effect of ouabain on resting [Na+]i in MD and cTAL cells. Addition of the Na-K-ATPase blocker ouabain to the bath did not significantly change resting [Na+]i in MD cells and had no effect on the MD [Na+]i dynamics in response to increased [NaCl]L. In contrast, bath ouabain significantly increased resting [Na+]i in cTAL cells (Fig. 4) up to the level found in MD cells (26.5 ± 6.7 mM, n = 6), and the 150 mM [NaCl]L-induced increases in [Na+]i (plateau at 61.3 ± 13.6 mM) were also similar to that found in MD cells. Importantly, the addition of 1 mM ouabain to the perfusate, in the continuous presence of bath ouabain, significantly increased resting [Na+]i in MD cells and caused a small but further elevation in cTAL [Na+]i. Thus MD cells (and also cTAL cells) possess an apical ouabain-sensitive Na+-efflux pathway. This is also supported by the representative recording in Fig. 3, where luminal ouabain almost completely blocked the recovery of [Na+]i that occurs on the return to the low-[NaCl]L perfusate. Thus these studies suggest that MD cells may possess an apically located, ouabain-sensitive colonic H-K-ATPase.


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Fig. 4.   Effects of bath or luminal ouabain (1 mM) on resting [Na+]i in MD and cTAL cells. Luminal administration of ouabain was done in the continuous presence of bath ouabain. Values are means ± SE; n = 6 each. ns, Not significant.*P < 0.05, compared with control (paired t-test).

Luminal K+-dependent pH recovery in MD cells. To functionally identify the presence of a colonic form of the H-K-ATPase at the apical membrane of MD cells, we used the experimental maneuver illustrated in Fig. 5A. Removal of external Na+, K+, and Cl- resulted in an MD intracellular acidification to ~pH 6.8, as monitored by the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In the continued absence of external Na+ and K+, no pHi recovery was detected. Readdition of only 5 mM K+ to the perfusate, however, resulted in a significant intracellular alkalinization, which is consistent with the presence of an apical H+/K+ exchange mechanism. Subsequent readdition of luminal Na+ caused a complete pHi-recovery, most likely due to apical Na+/H+ exchange. Figure 5B summarizes studies in MD cells where the addition of ouabain to the perfusate nearly abolished K-dependent pHi-recovery, whereas Sch-28080 had no effect.


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Fig. 5.   K+-dependent intracellular pH (pHi) recovery from an acid load in MD cells. A: representative recording. Acidification was achieved under K+- and Cl--free conditions by bilateral Na+ removal, which inhibits Na+/H+ exchanger activities. Readdition of 5 mM K+ to the lumen resulted in a partial pHi recovery. Subsequent luminal Na+ readdition caused a complete pHi recovery. For analysis of the H-K transport activity, we measured the initial rate of the K+-dependent pHi recovery (Delta pHi/Delta t) using PTI software. B: effects of luminal 10 µM Sch-28080 or 100 µM ouabain on the initial rate of K+-dependent pHi recovery. Values are means ± SE; n = 6 each. *P < 0.05, compared with control (t-test for 2 samples).

Effect of ouabain on the resting [Na+]i in the distal colon. To functionally identify the H-K-ATPase in rabbit MD cells, it was also necessary to characterize the pharmacological properties of the H-K-ATPase located in the rabbit distal colon. Using an approach that was similar to that used for the MD cells (Fig. 4), we measured changes in resting [Na+]i in response to luminal ouabain and Sch-28080 (Fig. 6). Bath ouabain increased intracellular [Na+]i, presumably due to the existence of Na-K-ATPase at the basolateral membrane. Importantly, luminal ouabain, but not Sch-28080, significantly increased resting [Na+]i, a response that was similar to the one observed in MD cells.


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Fig. 6.   Effects of bath and luminal ouabain (1 mM) or Sch-28080 (100 µM) on the resting sodium-binding benzofuran isophthalate (SBFI) fluorescent ratio in colonic crypt epithelial cells. Luminal administration of ouabain was performed in the continuous presence of bath ouabain to eliminate the possible effects of luminal ouabain on the basolateral Na-K-ATPase. Values are means ± SE; n = 6 each. *P < 0.05, compared with control using paired t-test.

Immunohistochemical localization of the Na-K-ATPase alpha 1-subunit. As illustrated in Fig. 7A, there was very little staining of the rabbit MD basolateral membrane with an antibody directed toward the Na-K-ATPase alpha 1-subunit. In contrast, adjacent cTAL cells and other surrounding nephron segments were strongly positive at the basolateral membrane.


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Fig. 7.   Immunofluorescence labeling of the Na-K-ATPase alpha 1-subunit in rabbit kidney A: distal tubular and cTAL cells are strongly positive at the basolateral membrane (green), whereas MD cells (arrow) are almost devoid of staining. G, glomerulus. Specific staining is absent when sections were incubated with the alpha 6F nonsecreting hybridoma supernatant (B). Nuclei are blue. Bar, 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies examined apical transport-related dynamics of [Na+]i in MD and cTAL cells. At normal physiological conditions prevailing at the end of the cTAL and at the MD, [NaCl]L is ~20-25 mM. Resting [Na+]i in MD cells, in the presence of 25 mM [NaCl]L and 150 mM NaCl in the bath (with the assumption that normal interstitial fluid [Na+]) was ~30 mM. This value is significantly higher than that found in other renal epithelial cells, measured by others with similar methods (17), and in adjacent cTAL cells in the present study. In addition, 25-150 mM [NaCl]L-induced elevations in MD [Na+]i were far greater than found in cTAL cells, further suggesting that there are fundamental differences between these two cell types in terms of [Na+]i regulation. Also of particular importance, the [NaCl]L-induced elevation in MD [Na+]i was sensitive to [NaCl]L over the range of 0 to 60 mM [NaCl]L (Fig. 2). This is exactly the same dynamic range of [NaCl]L that induces TGF responses (4). Thus parallel changes in [NaCl]L, [Na+]i, and TGF sensitivity suggest that [Na+]i plays a vital role in MD cell signaling.

It has long been recognized that apical Na+-2Cl--K+ cotransport is a major NaCl-entry pathway in MD cells. However, with the identification of an apical Na+/H+ exchanger, the relative contribution of these two Na+-entry pathways has not been determined. Using pharmacological inhibitors of cotransport (furosemide) and Na+/H+ exchange (HOE-642), we estimate that 80% of apical entry is through the cotransporter and ~20% is via the exchanger. Thus [Na+]i dynamics in MD cells reflects, to a very large extent, the kinetic properties and ion affinities of the cotransporter. The cotransporter is therefore responsible for the sigmoidal relationship between [NaCl]L and [Na+]i, with a plateau of [Na+]i at a [NaCl]L of 60 mM.

The representative recording in Fig. 1A demonstrates that the [NaCl]L-induced elevation in MD [Na+]i was reversible on return to low [NaCl]L, suggesting that there is an active Na+ extrusion mechanism. To rule out the possibility that passive efflux, through the Na+/H+ exchanger or Na+-2Cl--K+ cotransporter, was responsible for Na+-efflux, we assessed [Na+]i recovery in the presence of either the Na+-2Cl--K+ cotransport blocker furosemide or the Na+/H+ exchanger inhibitor HOE-642. Both agents failed to influence the recovery phase of [Na+]i when [NaCl]L was switched from 150 to 25 mM, thus providing a strong argument against passive efflux and suggesting that there is an active Na+ extrusion process. The most likely candidate for this Na+ efflux is the Na-K-ATPase, and, indeed, ouabain almost completely blocked the Na+ recovery phase (Fig. 3). However, as shown in Fig. 4, this effect of ouabain was unusual because it was effective from the luminal and not the basolateral side, where Na-K-ATPase is located. Because basolateral ouabain caused no significant change in resting [Na+]i in MD cells, but significantly increased resting [Na+]i in cTAL cells, the difference in [Na+]i dynamics between these two cell types can be explained by diminished Na-K-ATPase activity in MD cells.

Functional identification of an H-K-ATPase in MD cells and its localization to the apical membrane were evaluated by assessing ouabain-sensitive K+-dependent alkalinization on cell acidification imposed by prior bilateral Na+ removal. This experimental maneuver of cell acidification by removal of Na+ has been described for MD cells by our laboratory (15) and was also used in a previous study to characterize H-K-ATPase activity in the cortical collecting tubule (22). Addition of 5 mM luminal K+ resulted in MD cell pHi recovery, and this recovery was abolished by the addition of luminal ouabain (Fig. 5B). Because K+ at a concentration of 5 mM was used in the lumen during pH recovery, it is doubtful that there was significant proton efflux due to changes in cell membrane potential. Also, previous patch-clamp experiments (9) showed that the apical K+ channel in MD cells is almost completely inhibited by intracellular acidification to a pH of 6.8. Thus this K+-dependent pHi-recovery most likely occurred through the H-K-ATPase. Ouabain and Sch-28080 compounds are selective inhibitors for members of the Na-K/H-K-ATPase family (10, 12, 25). Pharmacological profiles of members of this family have been very well documented on the basis of studies in the rat or in expression systems using the cloned rat protein. The Na-K-ATPase is inhibited by ouabain but is insensitive to omeprazol, an inhibitor of the gastric H-K-ATPase, and its derivative Sch-28080. Gastric H-K-ATPase is inhibited by both omeprazol and Sch-28080, but not by ouabain, whereas nongastric H-K-ATPases are sensitive to ouabain, with certain isoforms showing sensitivity to very high concentrations of Sch-28080 (10, 12, 25). Drug sensitivity of the colonic H-K-ATPase in the rabbit has not yet been examined. Thus it was necessary to determine whether the rabbit colonic H-K-ATPase had a pharmacological profile similar to that found in other species such as the rat. Using an approach that was similar to what was used for the MD cell studies, we isolated and perfused individual crypts from distal colon and measured changes in resting [Na+]i in response to luminal ouabain and Sch-28080 (Fig. 6). The findings of these experiments suggest that the H-K-ATPase, located in the rabbit distal colon, has a pharmacological profile similar to that in the rat, i.e., sensitive to ouabain but not Sch-28080 and, like MD cells, able to transport Na+. Thus it can now be concluded that the ouabain-sensitive but Sch-28080-insensitive luminal K-dependent pHi recovery (Fig. 5B) indicates the presence of an H-K-ATPase located at the apical membrane of MD cells that belongs to the colonic isoform/subgroup. It is important to mention that all of the preparations used in these studies were from animals kept on a standard diet. This indicates constitutively active colonic H-K-ATPase in MD cells, in contrast to the low-K+-diet-induced isoforms in other nephron segments (7). Our functional studies are further supported by recent work (23) that localized the HKalpha 2c protein (a splice variant of rabbit colonic H-K-ATPase) at the apical membrane of MD cells using immunohistochemistry. Another important aspect of the present work is that it may shed some light on the functional properties of HKalpha 2c, because it has not yet been functionally expressed in heterologous systems and characterized.

Further evidence for the scarcity of basolateral Na-K-ATPase (alpha 1-subunit) in rabbit MD cells was obtained by immunohistochemistry. The staining of the MD basolateral membrane with an antibody directed toward Na-K-ATPase (Fig. 7A) was at the limit of detection. This is direct support for our functional studies, which demonstrated that bath addition of ouabain did not alter MD cell [Na+]. These results are also consistent with recent findings (1, 24) on the localization of the Na-K-ATPase alpha - and gamma -subunits in rat kidney. The gamma -subunit is a tissue-specific regulator of the functional activity of Na-K-ATPase in the kidney (1) and reduces the enzyme's affinities for its major physiological ligands, Na+ and K+. The gamma -subunit is highly expressed in MD cells at the basolateral membrane but, interestingly, is absent in adjacent cTAL cells (24). The colonic H-K-ATPase has been shown to assemble with different beta -subunits in heterologous systems (7, 10, 12, 25). The specific beta -subunit involved in apical Na(H)-K- ATPase in the MD remains to be determined. It should also be mentioned that we have performed a number of studies using this same MD transport-measuring experimental protocol and have never found evidence for a loss of cell polarity. Thus under these experimental conditions it is very unlikely that ischemia caused rearrangement of basolateral Na+ pumps to the apical membrane of the MD cells in the isolated and perfused cTAL preparation.

In summary, we identified and localized, to the apical membrane of MD cells, a colonic form of H-K-ATPase. Functional and immunological evidence indicates that these cells do not express other K-ATPases, suggesting that this colonic H-K-ATPase is the primary regulator of MD cell [Na+]i. The interesting observation that both Na+-influx and -efflux pathways are located at the apical membrane in these polarized epithelial cells offers a novel model of regulating cell [Na+]i, a Na+-recycling mechanism. MD cells are not likely to be primarily absorbing epithelia but rather function as sensor cells that detect changes in [NaCl]L. The interesting finding is that MD [Na+]i reflects changes in [NaCl]L between 0 and 60 mM, a finding that is consistent with the function of MD cells in TGF signaling. It should be mentioned that the HKalpha 2 isoform knockout mouse has recently been established (13), but TGF has yet to be examined. Further studies are necessary to determine whether the MD apical H-K-ATPase plays an important role in the sensor function of MD cells and in TGF signaling. We speculate that it is the presence of H-K-ATPase and lack of Na-K-ATPase that allows for such regulation of [Na+]i in MD cells and thus allows [Na+]i to reflect the luminal environment. We further suggest that the expression of particular forms of this ATPase family may be a generalized cellular mechanism that allows for regulation and setting of cell [Na+]i. Thus cells expressing the highly efficient Na-K-ATPase would have low [Na+]i, cells expressing H-K-ATPase may have higher [Na+]i, whereas cells expressing both forms may have an intermediate level of [Na+]i. Also, the recent findings of the inhibitory gamma -subunit suggests that there may be need for other Na+-efflux pathways during physiological inhibition of the Na-K-ATPase to maintain [Na+]i.

Thus K-ATPases may have a generalized function in helping to set cell [Na+]i. The fact that this pump works as an Na(H)-K-ATPase is consistent with earlier studies (6, 8) that examined Na+ transport via different isoforms of the nongastric H-K-ATPase in expression systems and also with more recent work (5, 16) using isolated apical membranes of rat distal colon. Wide distribution of HKalpha 2 protein in distal colon and nephron segments suggests that the colonic H-K-ATPase might also have a yet unrecognized physiological importance in Na+ transport and electrolyte homeostasis.


    ACKNOWLEDGEMENTS

We thank Dr. Strader (Schering-Plough Research Institute, Kenilworth, NJ) for providing the Sch-28080. The antibody alpha 6F was from the Developmental Studies Hybridoma Bank, University of Iowa.


    FOOTNOTES

We also thank Suzanne Randall, University of Alabama at Birmingham, Department of Surgery-Neurosurgery, for excellent help in tissue processing for immunohistochemistry.

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32032 (to P. D. Bell). J. Peti-Peterdi was a National Kidney Foundation Postdoctoral Fellow during these studies.

Address for reprint requests and other correspondence: J. Peti-Peterdi, 865 Sparks Ctr., 1720 Seventh Ave. South, Birmingham, AL 35294 (E-mail: petjan{at}uab.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 October 10, 2001; 10.1152/ajprenal.00251.2001

Received 15 August 2001; accepted in final form 4 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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