INVITED REVIEW
H+-K+-ATPases: regulation and role in pathophysiological states

Randi B. Silver and Manoocher Soleimani

Department of Physiology and Biophysics, Cornell University Medical College, New York, New York 10021; and the Department of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0585


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
THE HKAS: NEPHRON SEGMENT...
HKA: FUNCTIONAL STUDIES
HKA: INHIBITOR SENSITIVITY
HKA: ADAPTIVE REGULATION
REFERENCES

Molecular cloning experiments have identified the existence of two H+-K+-ATPases (HKAs), colonic and gastric. Recent functional and molecular studies indicate the presence of both transporters in the kidney, which are presumed to mediate the exchange of intracellular H+ for extracellular K+. On the basis of these studies, a picture is evolving that indicates differential regulation of HKAs at the molecular level in acid-base and electrolyte disorders. Of the two transporters, gastric HKA is expressed constitutively along the length of the collecting duct and is responsible for H+ secretion and K+ reabsorption under normal conditions and may be stimulated with acid-base perturbations and/or K+ depletion. This regulation may be species specific. To date there are no data to indicate that the colonic HKA (HKAc) plays a role in H+ secretion or K+ reabsorption under normal conditions. However, HKAc shows adaptive regulation in pathophysiological conditions such as K+ depletion, NaCl deficiency, and proximal renal tubular acidosis, suggesting an important role for this exchanger in potassium, HCO-3, and sodium (or chloride) reabsorption in disease states. The purpose of this review is to summarize recent functional and molecular studies on the regulation of HKAs in physiological and pathophysiological states. Possible signals responsible for regulation of HKAs in these conditions will be discussed. Furthermore, the role of these transporters in acid-base and electrolyte homeostasis will be evaluated in the context of genetically altered animals deficient in HKAc.

proton-potassium-adenosinetriphosphatase; kidney; potassium depletion; sodium depletion; acid-base homeostasis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
THE HKAS: NEPHRON SEGMENT...
HKA: FUNCTIONAL STUDIES
HKA: INHIBITOR SENSITIVITY
HKA: ADAPTIVE REGULATION
REFERENCES

ACTIVE PROTON SECRETION by the collecting duct (CD) is coupled in part to active K+ absorption via a membrane-bound ion exchanger, H+-K+-ATPase (HKA) (36, 81). The 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 (36, 59, 81) and will be referred to as gastric HKA (HKAg). A distinct but structurally related HKA is expressed in the distal colon, which mediates active K+ reabsorption (15). This colonic transporter as it relates to kidney will be called colonic HKA (HKAc) or nongastric HKA. Molecular studies indicate expression of both HKAg and HKAc in CD (1, 2). Functionally they are thought to be involved in H+ secretion and/or K+ reabsorption but appear to be regulated differentially. Furthermore, HKAc may also mediate the exchange of extracellular K+ for intracellular Na+ or NH+4 (3, 13), consistent with this transporter working on cation/potassium exchange mode.

In the following sections, we will discuss the distribution of this class of K+-dependent ATPases in the nephron, their functional characteristics, and their adaptive regulation under both physiological and pathophysiological conditions.


    THE HKAS: NEPHRON SEGMENT DISTRIBUTION
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ABSTRACT
INTRODUCTION
THE HKAS: NEPHRON SEGMENT...
HKA: FUNCTIONAL STUDIES
HKA: INHIBITOR SENSITIVITY
HKA: ADAPTIVE REGULATION
REFERENCES

Gastric HKA

K+-ATPase activity in kidney was first demonstrated over a decade ago by measuring K+-dependent ouabain-insensitive ATP hydrolysis in individually microdissected nephron segments from rat and rabbit (18, 25). Collectively these studies showed that under normal conditions there was significant K+-ATPase activity measured in segments from the distal nephron. The K+-ATPase activity was highest in the connecting tubule, lowest in the outer medullary collecting duct (OMCD), and intermediate in the cortical collecting duct (CCD). This K+-dependent ATPase activity was insensitive to ouabain and inhibited by vanadate, Sch-28080, and omeprazole, with the latter two being specific inhibitors of HKAg (41, 73). In addition, because there appeared to be a correlation between the magnitude of enzymatic activity and the percentage of intercalated cells (ICs) in a given nephron segment, it was proposed that this K+-ATPase activity might be specifically found in the ICs (18). This enzymatically identified Sch-28080-sensitive and ouabain-insensitive activity found in distal nephron from control rats, presumably represents what has since been labeled type I renal K+-ATPase (9). Consistent with this enzymatic finding was the functional demonstration in OMCD from K+-depleted rabbit of an active, proton-pumping and K+-absorptive mechanism that was inhibited by omeprazole (79) and Sch-28080 (80). This study (79) was the first direct demonstration of functional HKA activity and provided a mechanism whereby the OMCD could actively reabsorb K+ under hypokalemic conditions (79).

Immunoreactivity studies with monoclonal antibodies raised against the alpha -subunit of the hog HKAg revealed diffuse cytoplasmic staining in ICs in rat and rabbit CCD and OMCD (81). Double labeling experiments performed on rat OMCDs demonstrated positive staining for HKAg and the basolateral Cl-/HCO-3 exchanger, a band 3 type protein specific to the alpha -type IC (81). Another immunocytochemistry study in rat CD demonstrated that HKAg colocalized with H-ATPase in both alpha -type and beta -type ICs, suggesting an apical location for HKAg in alpha -type ICs and a basolateral location in beta -type ICs (7). Perfusion studies on isolated rabbit CCDs have shown that Sch-28080-sensitive HKA exists in beta -type ICs on the apical membrane (62, 78), which is in contrast to the immunohistochemical findings in rat CCD (7). This may reflect species-specific differences.

At the molecular level mRNA expression studies have shown that HKAg appears to be more widely expressed in the CD. In kidney from normal rats, in situ hybridization studies using a specific cRNA probe for the gastric alpha -subunit showed mRNA expression in all of the cells of the connecting tubule, CCD [principal cells (PCs) and ICs], and ICs of the OMCD outer stripe and inner medullary CD (IMCD) (1). In situ hybridization of HKA beta -subunit mRNA in normal rat kidney showed that this gene is expressed in ICs of the connecting tubule, CCD, and outer stripe of OMCD, PCs and ICs of the inner stripe of OMCD, and the IMCD (10).

Colonic HKA

Northern hybridization studies have demonstrated that mRNA for HKAc is expressed in kidney (15). Specifically, it has been identified in renal cortex (2, 19, 33, 56, 75) and in individual CCDs (70) of control rats, but the expression is much less compared with distal colon (15). RT-PCR analysis of mRNA encoding the alpha -subunit of HKAc revealed expression of this transporter in CD of rats with K+ depletion (45). In situ hybridization studies with an HKAc-specific alpha -subunit cRNA probe showed labeling in CCD and IMCD segments of rat kidney from control animals, with lower expression levels in OMCD (2). There was also detectable mRNA expression of HKAc in the thick ascending limb of Henle (TAL) (2). At the protein level, an antibody raised against a fusion protein encoded by the first 327 bp from the 5' end of the coding region of the alpha -subunit of HKAc recognized ~100-kDa apical membrane protein from the distal rat colon by Western blot (40). Using this same antibody to HKAc, a similarly sized protein was identified by Western blot analysis from renal outer medulla of K+-depleted rats (56). Immunocytochemical analysis with this protein localized HKAc to the apical membrane of the surface epithelial cells of the rat distal colon (40) and the PCs of OMCDs (56).

Other K+-ATPases

Recently, two other K+-dependent P-type ATPases, referred to as type II renal K+-ATPase and type III renal K+-ATPase (9), were enzymatically identified in microdissected rat nephron segments and appear to be differentially regulated (9, 83). Pharmacologically, these K+-dependent ATPase activities exhibit different sensitivity profiles to Sch-28080 and ouabain compared with the type I renal K+-ATPase, the Sch-28080-sensitive, ouabain-insensitive HKAg, and the Sch-28080-insensitive, ouabain-sensitive HKAc. Types II and III renal K+-ATPases have been shown to be moderately inhibited by both Sch-28080 and ouabain (9). Working on microdissected nephron segments from control rats, type II K+-ATPase activity was found in proximal tubule (PT) and TAL. With K+ depletion, however, this K+-ATPase activity disappeared in the PT and TAL. Under these hypokalemic conditions, K+-ATPase activity appeared in CCD which was not observed under control conditions. This type III renal K+-ATPase activity was characterized as having different sensitivities to Sch-28080 and ouabain compared with types I and II K+-ATPase (9, 83).

Figure 1A is a schematic diagram of the kidney as represented by a nephron and shows where the various renal K+-dependent ATPases are found in the kidney. This renal distribution is based on evidence derived from rat and rabbit using biochemical and functional assays. It should be realized that this analysis tells an incomplete story and overlooks species-specific differences. However, it serves as a reminder of how much more data need to be generated to better understand the role of these proteins in the kidney.



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Fig. 1.   H+-K+-ATPases (HKAs) and K+-dependent ATPases in the kidney: functional and molecular characterization. A: distribution of K+-dependent ATPases in the nephron based on inhibition by Sch-28080 and ouabain under control, K+-depleted, NaCl-depleted, and acidotic conditions. Unitary nephron representation of the kidney under four conditions: control, K+ depletion, NaCl depletion, and acidosis. Five K+-dependent ATPases are identified. PT, proximal tubule; TAL, thick ascending limb; DT, distal tubule; CCD, cortical collecting duct; and IMCD, inner medullary CD. B: distribution of HKAs in the nephron based on molecular characterization of HKAg and HKAc. The unitary nephron represents the distribution in the kidney. Distribution of these two HKAs is compared between control and K+ depletion. The difference in contrast between control and K+ depletion represents changes in expression.

The five K+-dependent renal K+-ATPases represented in Fig. 1A are as follows: the Sch-28080-sensitive and ouabain-insensitive HKA, the Sch-28080-insensitive and ouabain-sensitive HKA, the Sch-28080-sensitive and ouabain-insensitive K+-ATPase (type I), the Sch-28080-sensitive and ouabain-sensitive K+-ATPase (type II), and the Sch-28080-sensitive and ouabain-sensitive K+-ATPase (type III). The distribution of these K+-dependent ATPases is depicted under physiological and the following three pathophysiological conditions: K+ depletion, NaCl depletion, and acidosis.

Under control conditions, enzymatic studies have localized type I K+-ATPase to CCD and OMCD and type II K+-ATPase to proximal convoluted tubule (PCT) and TAL (9). Functional studies performed on control rabbit and rat tubules have demonstrated that Sch-28080-sensitive and ouabain-insensitive HKA exists all along the CD, including IMCD, OMCD, and CCD (5, 12, 27, 39, 60, 61, 63, 78). In isolated nephron segments from K+-depleted rat, type I K+-ATPase and type II K+-ATPase disappear, but type III K+-ATPase arises in CCD and OMCD (9). In K+-depleted tubules from rat and rabbit, Sch-28080-sensitive and ouabain-insensitive HKA has been demonstrated in CD from CCD to IMCD (5, 22, 36, 39, 40, 51, 74, 79, 80, 84). In NaCl depletion, functional data derived from rat CCD demonstrate the existence of Sch-28080-sensitive, ouabain-insensitive HKA and Sch-28080-insensitive, ouabain-sensitive HKA (60). Under conditions of acidosis there is functional evidence for the Sch-28080-sensitive and ouabain-insensitive HKA along the length of the CD (21, 63, 72).

Figure 1B represents the distribution of HKAg and HKAc under control and K+-depleted conditions based on the molecular characterization of these transporters. Differences in contrast between control and K+ depletion relate to the differential expression of these HKAs observed between these two states. For HKAg, mRNA expression of the alpha -subunit of the gastric pump is seen in CCD, OMCD, and IMCD (1). With K+ depletion, the signal in CCD is enhanced (36) and diminished in IMCD (49). In control conditions mRNA expression of the alpha -subunit of HKAc has been demonstrated in TAL, CCD, OMCD, and IMCD (2). In K+ depletion, the signal for HKAc is enhanced in CD (2, 49, 50) and remains the same in TAL (2).


    HKA: FUNCTIONAL STUDIES
TOP
ABSTRACT
INTRODUCTION
THE HKAS: NEPHRON SEGMENT...
HKA: FUNCTIONAL STUDIES
HKA: INHIBITOR SENSITIVITY
HKA: ADAPTIVE REGULATION
REFERENCES

Gastric HKA

HKAg will be functionally distinguished by its ability to be inhibited by specific blockers. HKAg is blocked by compounds such as vanadate (a P-type ATPase inhibitor) and the more specific inhibitors Sch-28080 and omeprazole. HKAg differs from its relative the Na+ pump by being insensitive to ouabain. In the distal tubule, it is believed that HKAg plays a role in both K+ reabsorption and acid secretion, as first suggested by numerous enzymatic studies that measured K+-dependent ATPase activity. These studies demonstrate that ouabain-insensitive K+-ATPase activity exists in distal nephron segments of rat and rabbit and is inhibited by vanadate and omeprazole and Sch-28080, strongly suggestive that this activity represents functional HKAg (18, 20, 25).

In addition to biochemical identification of HKAg activity in distal nephron, functional studies have also demonstrated its existence. The first functional study consistent with HKAg activity demonstrated active acidification and K+ reabsorption in isolated and perfused inner OMCD from K+-deprived rabbits (79). Luminal addition of omeprazole abolished this transport, suggesting an apical location for the H+/K+ exchange. Subsequent studies performed on microperfused distal tubules from K+-depleted rats showed similar findings where addition of Sch-28080 to the luminal perfusate reduced HCO-3 absorption (22, 49, 74). HKA was also found in the OMCD from K+-replete rabbits where microperfused OMCDs possessed Sch-28080-sensitive net flux of total CO2 (JtCO2) (3, 79). In another study using microperfused OMCDs from the OMCD outer (OMCDo) and inner (OMCDi) stripes of K+-replete rabbits, the rate of K+-dependent intracellular pH (pHi) recovery in response to an acute acid pulse was inhibited with luminal addition of Sch-28080 in the ICs, and OMCDi cells, but not in the PCs of the OMCDo (39). This K+-dependent rate of intracellular alkalinization was not affected by addition of the H+-ATPase inhibitor bafilomycin. In isolated and perfused OMCD and initial IMCD from normal rats, addition of Sch-28080 to the luminal perfusate decreased total CO2 absorption, implying an apical location for the HKA in these segments (27). These studies demonstrate the functional existence of Sch-28080-sensitive HKA under control and K+-depleted conditions in OMCD and IMCD from rat and rabbit.

Sch-28080-sensitive H+/K+ exchange has also been demonstrated in cultured mouse IMCD and OMCD cells (32, 52) and rat IMCD cells (35). When the mouse OMCD cells were maintained in low-potassium medium the K+-dependent pHi recovery rate from an imposed acid load was greater than the rate measured on cells maintained under normokalemic conditions (32). PCR analysis of these cultured mouse OMCD cells showed that both HKAg and HKAc were expressed. In cultured IMCD cells, Sch-28080-sensitive H+/K+ exchange has also been observed in response to an acute acid pulse and is thought to play a role in pHi regulation (35, 52).

In ICs from split-open CCDs from control rabbit and rat, K+-dependent pHi recovery rates were measured in response to an acute acid pulse that were Sch-28080 sensitive (10 µM) and ouabain insensitive (12, 60, 61, 63). In isolated and microperfused rat CCD (27) addition of Sch-28080 to the luminal perfusate did not appear to have any effect on JtCO2 absorption. However, in microperfused CCDs from chronically alkalotic rats, addition of Sch-28080 to the bath inhibited JtCO2 secretion, suggesting a basolateral location of HKA with alkalosis (27). These results differ from the results observed in microperfused rabbit CCD where an apical Sch-28080-sensitive HKA has been identified in beta -type ICs from control (78) and acidotic (63) rabbits. It should be emphasized that the Schering compound is membrane permeable, and as indicated (82), it may not be the most appropriate way of deciding sidedness. A charged membrane-impermeant version of the Sch-28080 compound is preferable for this purpose as described (82).

In the functional studies on CCD, the results primarily reflect the response of the ICs. The PCs, however, may also possess functional HKA. PCs from split-open rabbit CCD show K+-dependent pHi alkalinization in response to an acute acid pulse at rates comparable to those measured in ICs (61). The difference between the ICs and the PCs is in the sensitivity to Sch-28080; in the PCs, Sch-28080 does not inhibit the K+-dependent pHi recovery rate (61). Perhaps this difference in Sch-28080 sensitivity reflects another variant of HKA in PCs. Immunohistochemical localization of HKAg and HKAc in kidney should help to clarify this issue.

Colonic HKAc

The functional properties of the HKAc have been examined in various in vitro expression systems and are discussed in detail in the accompanying review article (32a). The inhibition of HKAc by ouabain has been studied in two different systems and requires special emphasis. In Xenopus oocytes, the alpha -subunit of HKAc was by coexpressed along with the beta -subunit of toad bladder HKA (14). This resulted in functional H+/K+(Rb+) exchange that was 50% inhibited in the presence of almost 1 mM ouabain and was insensitive to 500 µM Sch-28080 (14). In a different system, which measured K+-activated ATPase activity in apical membrane preparations enriched with endogenous colonic K+-ATPase, the Km for ouabain was reported to be 100 µM (17); representing almost an order of magnitude greater sensitivity to ouabain than that measured in the oocytes (14). These disparate results need to be reconciled using native intact tissue. Like the oocyte study there was no inhibition of K+-ATPase activity by high concentrations of Sch-28080 in the apical membrane preparations derived from colon (17).

Functional studies on native kidney tubules from control rats show that HKAc does not appear to be active under control conditions. In microperfused OMCD and terminal IMCD (IMCDt) from control rats, luminal addition of 1 mM ouabain has no effect on HCO-3 reabsorption, indicating that HKAc does not play an important role in H+ secretion and HCO-3 reabsorption under these conditions (49, 50). Similarly, in ICs studied in split-open CCD from rats maintained on a control diet, 1 mM ouabain had no effect on the rate of K+-dependent pHi recovery in response to an acute pulse of NH4Cl, whereas the addition of Sch-28080 inhibited much of this recovery (60). These functional studies support the notion that under normal conditions HKAc does not contribute in a significant way to acid secretion.

In addition to the ouabain-sensitive HKAc, another K+-ATPase has been described in colon that is insensitive to ouabain (1 mM), but is sensitive to vanadate, and localized to apical membrane of surface cells of rat distal colon (40). This suggests that two distinct K+-dependent ATPases may exist in colon: one that is ouabain sensitive and one that is ouabain insensitive. Immunocytochemical localization of the ouabain-insensitive colonic K+-ATPase in distal nephron showed nonspecific diffuse cytoplasmic staining in renal cortex and as mentioned earlier, apical staining of the PCs in OMCD, which was enhanced with K+ depletion (56).

Most recently a novel NH2-terminal splice variant of the rat HKAc alpha -subunit (HKA alpha 2b) has been cloned (37). The HKA alpha 2b is present in the cortex, outer medulla, and inner medulla and shows greater upregulation than the original alpha 2-subunit in K+ depletion (37). The renal cell distribution of HKA alpha 2b and its regulation in other pathophysiological states remains to be determined.


    HKA: INHIBITOR SENSITIVITY
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ABSTRACT
INTRODUCTION
THE HKAS: NEPHRON SEGMENT...
HKA: FUNCTIONAL STUDIES
HKA: INHIBITOR SENSITIVITY
HKA: ADAPTIVE REGULATION
REFERENCES

It has become apparent that variants of HKA have different sensitivities to Sch-28080 and/or ouabain, which may be used to distinguish them from each other. The inhibitory profile of HKAs in heterologous expression systems is discussed by Jaisser and Beggah in the accompanying article (32a). We therefore will only highlight the inhibitory profile of HKAs in native tissues.

The ouabain-insensitive HKAg is specifically inhibited by the hydrophobic, uncharged imidazopyridine, Sch-28080, which competes with K+ as determined in gradient-purified hog gastric microsomes (73). Inhibition of the gastric acid pump with Sch-28080 ranged in concentrations from 0.1 to 10 µM with half-maximal inhibition (i.e., IC50) of 0.15 µM at pH 6.5 (73). This is not dissimilar to the IC50 of 0.5 µM in the presence of 2.5 mM K+ reported for isolated CCDs and OMCDs from K+-depleted rats (16).

The discovery of a novel HKA from toad bladder with a Ki of 230 µM for Sch-28080 and a Ki of 25 µM for ouabain (34) led investigators to reevaluate the ouabain sensitivity of K+-ATPases in the distal nephron (83). The original enzymatic assays that identified K+-ATPase activity in mammalian distal nephron (18, 25) did so in the presence of ouabain to inhibit endogenous Na+ pump activity. A modification of the enzymatic assay was developed so that ouabain-sensitive Na+ pump activity could be distinguished from K+- and Sch-28080-dependent HKA activity. With this revised assay, Sch-28080 and ouabain-sensitive K+-ATPase activity was demonstrated in rat PT and TAL under control conditions (9, 83). The IC50 values were reported to be 1.7 µM for Sch-28080 and 6.4 µM for ouabain (9). This activity was distinctly different from the Sch-28080-sensitive and ouabain-insensitive K+-ATPase activity also measured in CD from these control rats (9). Furthermore, this study showed that under K+-depleted conditions an additional activity was measured in CD which is sensitive to both Sch-28080 and ouabain with an IC50 of 0.85 µM for Sch-28080 and an IC50 of 20 µM for ouabain. Although the molecular identity of the proteins associated with these activities is not known, it is possible that either inhibition profiles of the various types of HKA differ between heterologous expression systems and native tissue or alternatively novel isoforms account for the discrepancies.


    HKA: ADAPTIVE REGULATION
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ABSTRACT
INTRODUCTION
THE HKAS: NEPHRON SEGMENT...
HKA: FUNCTIONAL STUDIES
HKA: INHIBITOR SENSITIVITY
HKA: ADAPTIVE REGULATION
REFERENCES

Regulation of HKA has been studied in several pathophysiological states including K+ depletion, NaCl deficiency, acid-base perturbations, and in conditions associated with increased HCO-3 delivery to distal nephron.

K+ Depletion

K+ depletion is known to induce and maintain metabolic alkalosis in rats and humans (54, 58), unlike rabbits and dogs, which develop metabolic acidosis. The maintenance of metabolic alkalosis by K+ depletion is mainly due to increased reabsorption of HCO-3 (11, 50, 54, 58, 64) and secretion of NH+4 (29, 67) in the kidney. Segmental analysis of the rat nephron by micropuncture or microperfusion technique indicates that K+ depletion increases net HCO-3 reabsorption in the PCT (54, 64), distal convoluted tubule (DCT) (11), and OMCD (50). The contribution of HKAg or HKAc to the maintenance of metabolic alkalosis in K+ depletion will be discussed in this section.

Microperfusion experiments on OMCD from K+-depleted rabbits have shown that HKA contributes significantly to HCO-3 and K+ reabsorption (79, 80). Both the K+ and HCO-3 absorption were inhibited by addition of omeprazole and Sch-28080 (79, 80). As mentioned earlier, it has also been shown that the rate of bafilomycin-insensitive K+-dependent pHi recovery in response to an acute acid load is enhanced with K+ depletion in rabbit OMCDi (39). This enhanced rate of K+-dependent intracellular alkalinization was not completely inhibited with addition of Sch-28080, suggesting that another HKA may be expressed with K+ depletion (39). It is postulated that the increased HKA activity observed in rabbit OMCDi with K+ depletion is an attempt to maximize potassium reabsorption from the tubular fluid (36, 82).

In microperfusion experiments on distal tubules from K+-depleted rats which includes DCT, connecting tubule, and initial collecting tubule, addition of Sch-28080 to the luminal perfusate abolished net K+ reabsorption (51) and reduced HCO-3 absorption (22, 74). In CCDs from K+-depleted rabbits, addition of Sch-28080 to the lumen decreased the lumen to bath flux of 86Rb presumably mediated by HKA (84). Based on the inhibitory profile of HKA activity in these studies (sensitivity to omeprazole and Sch-28080), it has been proposed that HKAg is the transporter responsible for increased activity in K+ depletion (36, 82).

Recent molecular studies in K+-depleted rats present another intriguing aspect. In rats placed on a K+-deficient diet for 2 wk, Northern hybridization and in situ hybridization studies showed that HKAc mRNA was significantly upregulated (2, 19, 50, 75). Induction of HKAc is mostly evident in the medulla (2, 50, 75) but also shows moderate intensity in the cortex (50). In the medulla of K+-depleted animals upregulation of HKAc is evident in both outer and inner medulla (49, 50). This occurs as early as 72 h after the start of the K+-deficient diet and precedes the onset of hypokalemia (3), indicating that the signal is likely activated by intracellular K+ depletion. HKAg mRNA and/or protein abundance remains unchanged in the whole kidney (19, 38), shows mild upregulation in the cortex (36), and is decreased in the inner medulla (49) of K+-depleted rats. Interestingly, upregulation of HKAc in K+ depletion is significantly blocked in hypophysectomized rats, indicating that pituitary hormones could play an important role on HKAc regulation in K+ depletion (75).

Enzymatic studies have shown that a ouabain-insensitive, Sch-28080-sensitive K+-dependent ATPase activity is present in CCD and OMCD of normal rats, consistent with functional HKAg (9). However, in K+-depleted rats, this Sch-28080-sensitive K+-ATPase is replaced with a ouabain- and Sch-28080-sensitive K+-ATPase (9). To examine the functional role of HKAs in K+ depletion, HCO-3 reabsorption in rat OMCD was measured and correlated with mRNA expression for the HKAs (50). With K+ depletion, HKAc mRNA expression was increased ~30-fold in outer medulla and JtCO2 in OMCD was enhanced (50). In normal rats, HCO-3 reabsorption in OMCD was decreased in the presence of 10 µM Sch-28080 but remained unchanged in the presence of 1 mM ouabain in the perfusate (50). In K+-depleted rats, however, ouabain at 1 mM decreased HCO-3 reabsorption significantly (50). Although 10 µM Sch-28080 also decreased HCO-3 reabsorption, the inhibitory effects of Sch-28080 and ouabain were not additive (50). These results suggest that in K+ depletion, HKAc is induced and mediates increased HCO-3 reabsorption in OMCD and that in vivo HKAc is sensitive to both Sch-28080 and ouabain.

The role and regulation of HKAs in inner medulla of K+-depleted animals has also been studied. HCO-3 reabsorption in IMCDt was increased in K+ depletion due to enhanced Sch-28080-sensitive HKA activity (71). Recent molecular and functional studies in our laboratory demonstrates that with K+ depletion HKAg expression is decreased, whereas HKAc mRNA is heavily induced, in the IMCDt of rats and JtCO2 in IMCDt was increased in K+-depleted animals (49). In control rats, 1 mM ouabain added to the perfusate had no effect on HCO-3 reabsorption, whereas 10 µM Sch-28080 decreased HCO-3 reabsorption significantly (49). In K+-depleted rats, 1 mM ouabain or 10 µM Sch-28080 decreased HCO-3 reabsorption; however, the inhibitory effects of Sch-28080 and ouabain on JtCO2 were not additive (49). These results indicate that, like the OMCD, HKAg is suppressed, whereas HKAc is induced in K+ depletion and mediates increased HCO-3 reabsorption in IMCDt.

In K+ depletion, HKAc mRNA and activity is heavily induced in outer and inner medulla (49, 50), suggesting strongly that this transporter becomes predominantly responsible for K+-dependent bicarbonate transport in medullary CD under this condition. These studies further indicate that the expression and/or activity of the gastric-like HKA, which is the dominant HKA under normal conditions, is downregulated in K+ depletion. This genetic datum is further supported by the functional observations that only with K+ depletion is bicarbonate transport inhibited by ouabain, a property of HKAc not shared by HKAg in vitro. Given the conclusion from these genetic and functional data then, the further observation that the effects of Sch-28080 and ouabain were not additive suggests that these inhibitors were acting on the same transporter. The acquisition of Sch-28080 sensitivity by HKAc in vivo in K+ depletion is therefore a new finding but confirmatory of that of Buffin-Meyer et al. (9). Since this is at variance with in vitro data (14), further studies will be required to clarify this issue. It is plausible that a novel nongastric, noncolonic HKA, which is sensitive to both Sch-28080 and ouabain, is induced in K+ depletion and mediates increased HCO-3 reabsorption in the medullary CD.

It has been proposed and widely accepted that upregulation of HKA in K+ depletion is to conserve K+ (36, 82). This conclusion has been based on the assumption that in K+ depletion, there is a need to minimize the obligatory urinary K+ loss. To accomplish that objective, animals presumably upregulate their HKA to increase luminal K+ reabsorption (in exchange for H+). Several lines of evidence, however, indicate that hypokalemia may not be the dominant signal for upregulation of HKA. A closer look at K+-depleted rats shows abnormalities in the excretion of other electrolytes that precede hypokalemia and may be involved in upregulation of HKA. Two electrolyte abnormalities that occur in K+ depletion are increased urinary chloride (42-44) and ammonium (NH+4) excretion (29, 68). K+ depletion causes renal chloride wasting via suppression of the apical Na+-K+-2Cl- cotransporter and the apical Na+-Cl- cotransporter (4). Although suppression of chloride-absorbing transporters, which precedes the onset of hypokalemia and occurs as early as 2 days after starting the K+-depleted diet (4), decreases reabsorption of Na+ and Cl- in medullary TAL and DCT, respectively, only urinary chloride excretion is increased (4, 42, 43), indicating possible increased excretion of NH+4, as urinary Na+ excretion remains normal (42, 43). Recent studies in our laboratories in rats on K+ depletion diet for 6 days show increased NH+4 excretion as demonstrated by elevated urinary anion gap (3). These studies demonstrated that in IMCDt, which is the major site for NH+4 secretion, the HKAc is heavily induced in K+ depletion, whereas the HKAg expression is decreased. NH+4 secretion by IMCDt was found to be enhanced in K+ depletion and inhibited by ouabain but not Sch-28080 in perfusate. Induction of HKAc was closely associated with induction of the secretary potassium channel ROMK1 (3). These results are consistent with the possibility that the HKAc can function in [NH+4]i/[K+]o ATPase exchange mode, and in K+ depletion, HKAc replaces gastric-like HKA to mediate the secretion of NH+4 into the lumen facilitated by K+ recycling by ROMK1. Figure 2 is a speculative schematic diagram demonstrating the role of HKAc in HCO-3 reabsorption (and/or NH+4 secretion) in IMCDt of K+-depleted rats.



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Fig. 2.   A speculative schematic diagram demonstrating regulation of HKAs in IMCD of normal (A) and K+-depleted (B) rats. AE, anion exchanger; NBC, Na+-HCO-3 cotransporter; ca, carbonic anhydrase.

K+ depletion is associated with decreased renal Na+ excretion (30, 31, 42, 55). Given the suppression of the apical Na+- and Cl--absorbing cotransporters (4) and the subsequent increased delivery of Na+ to distal nephron segments, decreased Na+ excretion indicates compensatory reabsorption of Na+ in the more distal segments. The two possible mechanisms that could mediate the reabsorption of Na+ in the distal nephron segments in K+ depletion are Na+ channel and HKAc (with Na+ transported on K+ site). Indeed, Na+ has been shown to substitute for K+ on HKAg in vesicles prepared from pig gastric mucosa (67). Moreover, Na+ showed competition with K+ on the Sch-28080-sensitive HKA in CCD of potassium-restricted rabbits (84). Functional studies in OMCD of K+-depleted rats, however, demonstrated that in the absence of K+ but presence of Na+ in perfusate, the HKA-mediated, Sch-28080- and ouabain-sensitive HCO-3 reabsorption is abolished (50), indicating that Na+ cannot substitute for K+ on rat HKAc. The only other possibility for increased Na+ reabsorption in distal nephron would be via the Na+ channel, which is known to operate electrogenically in exchange for K+. Induction of ROMK (the secretory K+ channels in CCD and OMCD) in IMCDt of K+-depleted rat (3) could result in K+ secretion with subsequent hyperpolarization of the luminal membrane. It is therefore plausible that induction of ROMK1 in K+ depletion can provide the electrogenic driving force for Na+ reabsorption through the amiloride-sensitive Na+ channel which is expressed in IMCDt cells. Electrophysiological studies will be needed to examine the Na+ channel activity in IMCDt in K+-depleted animals. It is worth mentioning that some of the Na+-retaining effect of K+ depletion could occur at the level of the PT and be mediated via enhanced luminal Na+/H+ exchanger and basolateral Na+-3HCO-3 cotransporter (64).

NaCl Deficiency

A recent study examined regulation of HKAs in NaCl deficiency (60). These experiments tested the hypothesis that chronic NaCl deprivation increases HKA function in rat ICs. Rat were placed on a NaCl-restricted diet for 10-14 days and HKA function was measured in ICs from CCDs using ratiometric fluorescence imaging of the pHi indicator, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The BCECF-loaded, split tubule preparation was used in combination with dual-excitation digital imaging to study H+/K+ exchange in ICs in response to an acute acid load. The results indicate that maintaining rats on a NaCl-restricted diet induces a ouabain-sensitive H+/K+ exchanger in the ICs which is not observed under control conditions (60). Only a Sch-28080-sensitive (10 µM) H+/K+ exchanger is measurable under control conditions (60). With chronic NaCl deficiency both the Sch-28080- and the ouabain-sensitive H+/K+ exchangers contribute equally to the K+-dependent pHi recovery rate after an acute acid pulse. The functional appearance of another H+/K+ exchanger that is insensitive to 10 µM Sch-28080 but is sensitive to ouabain is depicted in Fig. 1A. The net effect is an increased rate of K+-dependent pHi recovery from an acid load in the low-NaCl ICs compared with control ICs. Induction of the ouabain-sensitive HKA does not appear to be mediated by changes in plasma acid-base or K+ status, as serum K+ and acid-base parameters in NaCl-depleted rats are not different from that of rats on normal diet. Furthermore, upregulation of ouabain-sensitive HKA is not mediated via increased aldosterone, as rats injected with aldosterone for comparable period do not demonstrate any noticeable changes in HKA function compared with controls.

The variant of the ouabain-sensitive HKA, induced in the ICs with NaCl restriction, remains to be determined. Although the pharmacological profile of this exchanger has not yet been fully characterized, 1 mM ouabain appeared to inhibit H+/K+ exchange. Northern hybridization studies demonstrate an approximate fourfold increase in HKAc alpha -subunit mRNA expression in the renal cortices of rats on a NaCl-restricted diet (77). The HKAg mRNA expression remains unchanged in NaCl deficiency. This is similar to the results of Sangan et al. (56), who also saw an increase in the mRNA level for HKAc in renal cortex with NaCl depletion.

What is the physiological significance of increased HKA function under conditions of NaCl restriction? In control rats under normal conditions, the reabsorption of Na+ is minimal in the CD due to a dearth of Na+ channels in the PCs; however, under conditions of NaCl restriction with the resultant elevated plasma aldosterone levels, there is an abundance of apical Na+ channels in the PCs (26, 53). The higher density of Na+ channels as well as increased channel activity under these conditions leads to increased reabsorption of Na+ from the tubular fluid as demonstrated in isolated microperfused rabbit and rat cortical collecting tubules (57, 68). It is believed that the increase in the PC apical membrane Na+ permeability results in a more favorable electrical driving force for secretion of K+ and leads to a coupling between Na+ reabsorption and K+ secretion (53, 66). NaCl restriction is also known to increase Cl- reabsorption in the CD independent of plasma Cl- concentration, filtered Cl- load, or Cl- delivery to the CD (23).

It is speculated that the enhanced H+/K+ exchange observed with NaCl restriction may serve to reabsorb the K+ secreted in the neighboring PC. Figure 3 is a representative model of CCD with the two primary cell types: the Na+-transporting PCs, and the ICs represented by the beta -type and alpha -type. In the bicarbonate secreting beta -type IC model there is an apical Cl-/HCO-3 exchanger and HKA and a basolateral H+-ATPase. The alpha -type IC is modeled with an apical H+-ATPase and HKA and a disulfonic stilbene-sensitive Cl-/HCO-3 exchanger. The basolateral membrane in both IC sub-types has a Cl- and K+ conductance (47). It is postulated that under conditions of NaCl restriction, enhanced reabsorption of Na+ via the PCs leads to increased excretion of K+ into the tubular lumen. This secreted K+ could then be reabsorbed via the HKA in the neighboring alpha -type or beta -type IC. In addition to the K+ reabsorption via the apical HKA, in the beta -type IC there may be concomitant exchange of luminal Cl- for HCO-3, so that the net effect of these coordinated processes is reabsorption of Na+, K+, and Cl- and secretion of H+ and HCO-3. Therefore the increased abundance of HKA in NaCl depletion may serve to promote net NaCl reabsorption by this tubule segment, rather than H+ for K+ exchange.


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Fig. 3.   Model of transporters in the principal cell and alpha - and beta -type intercalated cells of CCD as they relate under conditions of NaCl depletion. SA, stretch activated.

HKAc has been shown to mediate the exchange of intracellular Na+ for extracellular K+ (13). Under conditions of NaCl depletion, it is unlikely that the ouabain-sensitive, Sch-28080-insensitive H+/K+ exchanger is operating in this mode for urinary Na+ excretion rate is decreased under these conditions, and there is no evidence of an acid-base perturbation (60). It is plausible that the ouabain-sensitive, Sch-28080-insensitive H+/K+ exchanger could operate in [Na+]i/[K+]o exchange mode in sodium-loaded states, thereby enhancing sodium excretion. Studies examining regulation of H+/K+ exchange under salt-loaded states should help to answer this question.

Acid-Base Disorders

The role and regulation of HKAs have also been examined in various acid-base disorders.

Respiratory acidosis or alkalosis. Regulation of HKAg and HKAc in respiratory acidosis or alkalosis have been studied by functional and molecular methods. A recent study measuring Sch-28080-sensitive enzymatic activity in distal nephron showed an enhancement of HKA activity with chronic hypercapnia in rat CCD and OMCD and an inhibition of this activity with chronic hypocapnia (21). The effect of acute respiratory acidosis on HKA activity was examined in rabbit CCD by exposing the tubules to 10% CO2 (85). These studies demonstrated that the Sch-28080-sensitive net HCO-3 reabsorption increased in the presence of 10% CO2, consistent with stimulation of HKAg (85). Northern hybridization studies have shown an upregulation of HKAc expression in chronic respiratory acidosis (28).

Metabolic acidosis. Metabolic acidosis is associated with augmentation of at least two HCO-3-reabsorbing processes: PT NHE-3 (65) and CD H+-ATPase (24). Induction of metabolic acidosis in rats by addition of NH4Cl in their drinking water resulted in increased NHE-3 activity and protein abundance in PT cells and increased H+-ATPase activity and redistribution (from cytosol to luminal membrane) in the CD. A recent study examined regulation of HKAs (gastric and colonic) in metabolic acidosis (19). Rats were placed on NH4Cl (0.3 M added to their drinking water) for 4 days and studied for the expression of renal HKAs. Northern hybridization studies revealed that both HKAg and HKAc mRNA remained unchanged in kidneys of rats with metabolic acidosis (19). In ICs from CCDs dissected from rabbits induced with chronic metabolic acidosis, the Sch-28080-sensitive HKA functional activity, assessed as the rate of K+-dependent pHi recovery in response to an acute acid pulse, was greater in ICs from the acidotic animals compared with controls (63).

In microperfused IMCDt from chronically acidotic rats, luminal addition of Sch-28080 reduced JtCO2 by 50% consistent with increased HKA (72). Luminal addition of bafilomycin had no effect on JtCO2 in IMCDt segment from acidotic rats. Like the study on OMCDi and OMCDo (39) these results also suggest that another HKA variant which is not inhibited by 10 mM Sch-28080 may be functionally expressed in the IMCD with chronic metabolic acidosis.

Proximal tubular acidosis. Recent studies have examined regulation of HKAs in conditions associated with increased delivery of HCO-3 to the distal nephron. In rats subjected to renal ischemia for 30 min, the expression and activity of the luminal Na+/H+ exchanger NHE-3 decreased 12 h after reperfusion, consistent with decreased reabsorption of HCO-3 in PT (76). Suppression of NHE-3 was associated with an approximately eightfold increase in HKAc mRNA in renal cortex 12 h after reperfusion (76). This raises the possibility that increased delivery of HCO-3 to the distal nephron, resulting from suppression of NHE-3 in the PT, could increase HKAc expression. To test this hypothesis, the effect of acetazolamide, a potent inhibitor of HCO-3 reabsorption in PT, on HKAc expression was studied. Rats injected with acetazolamide for 24 h showed the serum acid-base profile of hyperchloremic metabolic acidosis consistent with proximal renal tubular acidosis (RTA). Northern hybridization analysis showed an approximately fourfold increase in HKAc mRNA after 24 h of acetazolamide treatment (76). Unlike K+ depletion, hypophysectomy had no effect on HKAc upregulation in acetazolamide-treated rats (personal observation), indicating that the signal mediating upregulation of HKAc in K+ depletion is distinct from that in proximal RTA. HKAg expression in the kidney remained unchanged in renal ischemic reperfusion injury or in animals treated with acetazolamide (76). It is concluded that increased expression of HKAc may be vital to acid base homeostasis in early phase of acute ischemic renal failure by increasing HCO-3 reabsorption in distal nephron. It is intriguing to further postulate that increased expression of HKAc in response to acetazolamide may increase reabsorption of HCO-3 in distal nephron and reduce the impact of acetazolamide on HCO-3 loss.

The presence of type II K+-ATPase in the PT (9) raises the possibility that this transporter might contribute to H+ secretion and HCO-3 reabsorption in this nephron segment. It would therefore be possible, although speculative, that diminished activity of this transporter in certain pathophysiological conditions could lead to decreased HCO-3 reabsorption, with subsequent generation of proximal PTA.

HKA in wild-type and genetically engineered HKA-deficient mouse. The role of HKAs in H+ secretion or HCO-3 reabsorption in mouse kidney CD was recently studied in our laboratories (48). These studies show that in microperfused OMCD and IMCDt of the normal mouse, about two-thirds of net HCO-3 transport is mediated by a process inhibitable by Sch-28080 and not by ouabain in the lumen (48). Northern hybridizations in outer medulla and inner medulla showed high and moderate level of expression for HKAg, respectively (48). HKAc expression was not detectable. The functional data as they relate to the isoforms of HKA are consistent with an important role for HKAg but not HKAc in HCO-3 reabsorption in normal mouse medullary CD.

To better understand the role of HKAc in acid-base and electrolyte homeostasis, gene targeting technology was used to generate mice lacking the HKAc alpha -subunit. The strategy toward this end was to generate disruption of the alpha -subunit of the HKAc. A targeting vector consisting of a portion of genomic HKAc and a neo gene (to select for neomycin-resistance) was utilized to generate HKAc-deficient mice. Northern hybridization of HKAc mRNA in the distal colon of HKAc-deficient mice verified disruption of HKAc alpha -subunit (46). Serum electrolyte and acid-base profiles were normal in HKAc-deficient animals (46), consistent with the absence of a significant role for HKAc in HCO-3 reabsorption or K+ reabsorption under normal conditions. Wild-type mice on K+-deficient diet for 18 days showed increased expression of HKAc in their kidneys, whereas HKAc-deficient mice on the same diet did not show any detectable mRNA levels. HKAc mRNA levels in the distal colon of wild-type (a ~4.2-kb message) or deficient (a diffuse 6.2-kb message) animals remained unchanged in K+ depletion (46). HKAc-deficient mice placed on K+-deficient diet did not show statistically significant increased urinary excretion of Na+ or K+ compared with the wild type (46). However, fecal K+ excretion was greater in HKAc-deficient mice on normal or K+-deficient diet, indicating an important role for this transporter in K+ reabsorption in the colon. Acid-base profile of HKAc-deficient animals on normal or K+-deficient diet was not different from that of wild-type animals.

Comparable plasma sodium concentrations and urinary sodium excretion rates in HKAc-deficient and wild-type mice on K+-depleted diet (46) indicate that HKAc does not play a direct role in Na+ homeostasis or that compensatory mechanisms supervene. This by inference suggests that, at least in K+ depletion, HKAc in vivo does not operate on Nai/Ko or Ki/Nao exchange mode, as urine Na+ remained unaltered in HKAc-deficient mice on K+-depleted diet (46).

The absence of a significant difference between plasma potassium concentrations and urinary potassium excretion rates in HKAc-deficient and wild-type mice on K+-depleted diet (46) raises the possibility that HKAc upregulation in K+-depleted does not result in net K+ reabsorption. Although this possibility is in contrast to the accepted belief in the literature, it is in agreement with the hypothesis proposed in this article (see HKA: ADAPTIVE REGULATION, above) that HKAc induction in the medullary CD might result in recycling of the K+ secreted by ROMK (3) rather than net K+ reabsorption. Further studies on ROMK expression in the medullary CD of wild-type and HKAc-deficient mice on K+-depleted diet might help answer some of these questions. Additionally, one also has to consider species specificity of the effect of K+ depletion in acid-base balance. Whether mouse develops metabolic alkalosis in response to K+ depletion, similar to rat, or develops metabolic acidosis, similar to dog, remains unknown. Long-term studies (>4 wk of K+ deprivation) will help answer this question.

Interestingly, HKAg mRNA levels in the kidney were decreased in HKAc-deficient mice compared with control and were reduced further in K+ depletion (G. Shull, personal communication). Decreased HKAg mRNA in the kidneys of HKAc-deficient animals is interesting and suggests possible coordinated regulation of these two transporters at transcription level. Decreased expression of renal HKAg in K+-depleted wild-type mouse are consonant with those in rat demonstrating a switch from HKAg to HKAc in K+ depletion (49, 50).

In conclusion, HKAs are expressed in renal CDs and play an important role in acid-base and electrolyte homeostasis, with HKAg playing an important role under normal conditions, whereas HKAc shows activation in certain pathophysiological states. Future studies in genetically engineered mice deficient in HKAg or HKAc should answer questions regarding the role of these two transporters in electrolyte and acid-base homeostasis. Specifically, HCO-3 reabsorption, K+ secretion and reabsorption, and Na+ reabsorption in the CD of HKAg or HKAc-deficient animals need to be measured in various pathophysiological conditions, then correlated with the expression and activity of HCO-3-reabsorbing transporters, K+ channels, and Na+ channels, respectively. Comparison of these results with those of wild-type animals under similar conditions should shed more light on the role of these two transporters in acid-base and electrolyte homeostasis in vivo.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45828 (to R. B. Silver) and DK-46789 and DK-52821 (to M. Soleimani), by grants from the Underhill and Wild Wings Foundations (to R. B. Silver), and by grants from Dialysis Clinic Incorporated (to M. Soleimani).


    FOOTNOTES

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).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
THE HKAS: NEPHRON SEGMENT...
HKA: FUNCTIONAL STUDIES
HKA: INHIBITOR SENSITIVITY
HKA: ADAPTIVE REGULATION
REFERENCES

1.   Ahn, K. Y., and B. C. Kone. Expression and cellular localization of mRNA encoding the "gastric" isoform of H+-K+-ATPase alpha -subunit in rat kidney. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F99-F109, 1995[Abstract/Free Full Text].

2.   Ahn, K. Y., K. Y. Park, K. K. Kim, and B. C. Kone. Chronic hypokalemia enhances expression of the H+-K+-ATPase alpha 2- subunit gene in renal medulla. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F314-F321, 1996[Abstract/Free Full Text].

3.  Amlal, H., S. Nakamura, J. Galla, and M. Soleimani. Colonic H+-K+-ATPase mediates NH+4 secretion in inner medullary collecting duct in K+-depletion (Abstract). J. Am. Soc. Nephrol. In press.

4.   Amlal, H., Z. Wang, and M. Soleimani. Potassium depletion downregulates chloride-absorbing transporters in rat kidney. J. Clin. Invest. 101: 1045-1054, 1998[Abstract/Free Full Text].

5.   Armitage, F. E., and C. S. Wingo. Luminal acidification in K-replete OMCDi: contributions of H-K ATPase and bafilomycin-A1-sensitive H-ATPase. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F450-F458, 1994[Abstract/Free Full Text].

6.   Baird, N. R., H. Zaun, Z. Wang, and M. Soleimani. The effect of DOCA and potassium depletion on mRNA expression of ROMK isoforms (Abstract). J. Am. Soc. Nephrol. 8: 28, 1997.

7.   Bastani, B. Colocalization of H-ATPase and H-K ATPase immunoreactivity in the rat kidney. J. Am. Soc. Nephrol. 5: 1476-1482, 1995[Abstract].

8.   Bengele, H, E. R. McNamara, J. H. Schwartz, and E. A. Alexander. Acidification adaptation along the inner medullary collecting duct. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F1155-F1159, 1988[Abstract/Free Full Text].

9.   Buffin-Meyer, B., M. Younes-Ibrahim, C. Barlet-Bas, L. Chevel, S. Marsy, and A. Doucet. K depletion modifies properties of SCH-28080-sensitive K-ATPase in rat collecting duct. Am. J. Physiol. 272 (Renal Physiol. 41): F124-F131, 1997[Abstract/Free Full Text].

10.   Campbell-Thompson, M. L., J. W. Verlander, K. A. Curran, W. G. Campbell, B. D. Cain, C. S. Wingo, and J. E. McGuigan. In situ hybridization of H-K- ATPase beta -subunit mRNA in rat and rabbit kidney. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F345-F354, 1995[Abstract/Free Full Text].

11.   Capasso, G., P. Jaeger, G. Giebisch, V. Guckian, and G. Malnic. Renal bicarbonate reabsorption. II. Distal tubule load dependence and effect of hypokalemia. J. Clin. Invest. 80: 409-414, 1987[Medline].

12.   Constantinescu, A., R. B. Silver, and L. M. Satlin. H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct. Am. J. Physiol. 272 (Renal Physiol. 41): F167-F177, 1997[Abstract/Free Full Text].

13.  Cougnon, M., P. Bouyer., and F. Jaisser. Does the colonic H,K-ATPase also act as an Na,K-ATPase. Proc. Natl. Acad. Sci. USA. In press.

14.   Cougnon, M., G. Planelles, and F. Jaisser. The rat distal colon P-ATPase a subunit encodes a ouabain-sensitive H+,K+-ATPase. J. Biol. Chem. 267: 13740-13748, 1996[Abstract/Free Full Text].

15.   Crowson, M. S., and G. E. Shull. Isolation and characterization of a cDNA encoding the putative distal colon H+,K+-ATPase: similarity of deduced amino acid sequence to gastric H+,K+-ATPase and Na+,K+-ATPase and messenger RNA expression in distal colon, kidney and uterus. J. Biol. Chem. 267: 13740-13748, 1992[Abstract/Free Full Text].

16.   Cheval, L., C. Bartlet-Bas, C. Khodouri, E. Feraille, S. Marsy, and A. Doucet. K-ATPase-mediated Rb+ transport in rat collecting tubule: modulation during K+ deprivation. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F800-F805, 1991[Abstract/Free Full Text].

17.   Del Castillo, J. R., V. M. Rajendran, and H. J. Binder. Apical membrane localization of ouabain-sensitive K+ activated ATPase activities in rat distal colon. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G1005-G1011, 1991[Abstract/Free Full Text].

18.   Doucet, A., and S. Marsy. Characterization of K-ATPase in ion transport in the kidney. Am. J. Physiol. 252 (Renal Fluid Electrolyte Physiol. 21): F418-F423, 1987.

19.   DuBose, T. D., J. Codina, A. Burges, and T. A. Pressley. Regulation of H+-K+- ATPase expression in kidney. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F500-F507, 1995[Abstract/Free Full Text].

20.   Eiam-Ong, S., N. A. Kurtzman, and S. Sabatini. Regulation of collecting tubule adenosine triphosphates by aldosterone and potassium. J. Clin. Invest. 91: 2385-2392, 1992.

21.   Eiam-Ong, S., M. E. Laski, N. A. Kurtzman, and S. Sabatini. Effect of respiratory acidosis and respiratory alkalosis on renal transport enzymes. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F390-F399, 1994[Abstract/Free Full Text].

22.   Fernandez, R., M. J. Lopes, R. F. de Lira, W. F. Dantas, E. J. Cragoe, Jr., and G. Malnic. Mechanism of acidification along cortical distal tubule of the rat. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F218-F226, 1994[Abstract/Free Full Text].

23.   Galla, J. H., D. N. Bonduris, K. A. Kirk, and R. G. Luke. Effect of dietary NaCl on chloride uptake in rat collecting duct. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F454-F459, 1986[Medline].

24.   Gluck, S., and R. Nelson. The role of V-ATPase in renal epithelial H+ transport. J. Exp. Biol. 172: 205-218, 1992[Free Full Text].

25.   Garg, L. C., and N. Narang. Ouabain-insensitive K-ATPase in distal nephron segments of the rabbit. J. Clin. Invest. 81: 1204-1208, 1988[Medline].

26.   Garty, H., and L. G. Palmer. Epithelial sodium channels: function, structure, and regulation. Physiol. Rev. 77: 359-396, 1997[Abstract/Free Full Text].

27.   Gifford, J. D., L. Rome, and J. H. Galla. H+-K+-ATPase activity in rat collecting duct segments. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F692-F695, 1992[Abstract/Free Full Text].

28.   Gifford, J. D., M. W. Ware, S. Crowson, and G. E. Shull. Expression of a putative rat distal colonic H,K ATPase mRNA in rat kidney: effect of respiratory acidosis (Abstract). J. Am. Soc. Nephrol. 2: 700, 1991.

29.   Good, D. W. Active absorption of NH+4 by rat medullary thick ascending limb: inhibition by potassium. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F78-F87, 1988[Abstract/Free Full Text].

30.   Gopal Krishna, G., P. Chusid, and R. Hoeldtke. Mild potassium depletion provokes renal sodium retention. J. Lab. Clin. Med. 109: 724-730, 1987[Medline].

31.   Gopal Krishna, G., and S. C. Kapoor. Potassium supplementation ameliorates mineralocorticoid-induced sodium retention. Kidney Int. 43: 1097-1103, 1993[Medline].

32.   Guntupalli, J., O. Macaulay, S. Wall, R. Alpern, and T. D. DuBose, Jr. Adaptation to low-K+ media increases H+-K+-ATPase but not H+-ATPase mediated pHi recovery in OMCDi cells. Am. J. Physiol. 273 (Cell Physiol. 42): C558-C571, 1997[Abstract/Free Full Text].

32a.   Jaisser, F., and A. T. Beggah. The nongastric H+-K+-ATPases: molecular and functional properties. Am. J. Physiol. 276 (Renal Physiol. 45): F812-F824, 1999[Abstract/Free Full Text].

33.   Jaisser, F., B. Escoubet, N. Coutry, E. Eugene, J. P. Bonvalet, and N. Farman. Differential regulation of putative K+-ATPase by low-K+ diet and corticosteroids in rat distal colon and kidney. Am. J. Physiol. 270 (Cell Physiol. 39): C679-C687, 1996[Abstract/Free Full Text].

34.   Jaisser, F., J. D. Horisberger, K. Geering, and B. C. Rossier. Mechanism of urinary K and H excretion: primary structure and functional expression of a novel H,K-ATPase. J. Cell Biol. 6: 1421-1431, 1993.

35.   Kleinman, J. G., P. Tipnis, and R. Pscheidt. H+-K+-ATPase of rat inner medullary collecting duct in primary culture. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F698-F704, 1993[Abstract/Free Full Text].

36.   Kone, B. C. Renal H,K-ATPase: structure, function, and regulation. Miner. Electrolyte Metab. 22: 349-365, 1996[Medline].

37.   Kone, B. C, and S. C. Higham. A novel N-terminal splice variant of the rat H+-K+-ATPase alpha 2 subunit. J. Biol. Chem. 273: 2543-2552, 1998[Abstract/Free Full Text].

38.   Kraut, J. A., J. Hiura, M. Besancon, A. Smolka, G. Sachs, and D. Scott. Effect of hypokalemia on the abundance of HKalpha 1 and HKalpha 2 protein in the rat kidney. Am. J. Physiol. 272 (Renal Physiol. 41): F744-F750, 1997[Abstract/Free Full Text].

39.   Kuwahara, M., W. J. Fu, and F. Marumo. Functional activity of H-K-ATPase in individual cells of OMCD: localization and effect of K+ depletion. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F116-F122, 1996[Abstract/Free Full Text].

40.   Lee, J., V. M. Rajendran, A. S. Mann, M. Kashgarian, and H. J. Binder. Functional expression and segmental localization of rat colonic K-ATPase. J. Clin. Invest. 96: 2002-2008, 1995[Medline].

41.   Long, J. F., P. J. Chiu, M. J. Derelanko, and M. Steinberg. Gastric antisecretory and cytoprotective activities of SCH 28080. J. Pharmacol. Exp. Ther. 226: 114-120, 1983[Abstract].

42.   Luke, R. G., and H. Levtin. Impaired renal conservation of chloride and the acid base changes associated with potassium depletion in the rat. Clin. Sci. (Colch.) 32: 511-526, 1967[Medline].

43.   Luke, R. G., F. S. Wright, N. Fowler, M. Kashgarian, and G. H. Giebisch. Effect of potassium depletion on renal tubular chloride transport in the rat. Kidney Int. 14: 414-427, 1978[Medline].

44.   Mannitius, A., H. Levitin, D. Beck, and F. H. Epstein. On the mechanism of impairment of renal concentrating ability in potassium deficiency. J. Clin. Invest. 39: 684-692, 1960.

45.   Marsy, S., J. M. Elalouf, and A. Doucet. Quantitative RT-PCR analysis of mRNAs encoding a colonic putative H,K-ATPase a subunit along the rat nephron: effect of K+ depletion. Pflügers Arch. 432: 494-500, 1996[Medline].

46.   Meneton, P., P. J. Schultheis, G. Jeannettem, M. L. Nieman, L. L. Clarke, J. J. Duffy, T. Doetschman, J. N. Lorenz, and G. Shull. Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice. J. Clin. Invest. 101: 536-542, 1998[Abstract/Free Full Text].

47.   Muto, S., K. Yasoshima, K. Yoshitomi, and Y. Asano. Electrophysiological identification of a- and b- intercalated cells and their distribution along the rabbit distal nephron segments. J. Clin. Invest. 86: 1829-1839, 1990[Medline].

48.   Nakamura, S., H. Amlal, M. J. Soleimani, and J. Galla. Mechanism of HCO-3 reabsorption in the perfused mouse OMCD and IMCDt (Abstract). J. Am. Soc. Nephrol. 8: 8, 1997.

49.   Nakamura, S., H. Amlal, J. Galla, and M. Soleimani. Colonic H+-K+-ATPase is induced and mediates HCO-3 reabsorption in IMCDt in potassium depletion. Kidney Int. 54: 1233-1239, 1998[Medline].

50.   Nakamura, S., Z. Wang, J. H. Galla, and M. Soleimani. Potassium depletion increases HCO-3 reabsorption in outer medullary collecting duct by activation of colonic H-K-ATPase. Am. J. Physiol. 274 (Renal Physiol. 43): F687-F692, 1998[Abstract/Free Full Text].

51.   Okusa, M. D., R. J. Unwin, H. Velazquez, and G. Giebisch. Active potassium absorption by the renal distal tubule. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F488-F493, 1992[Abstract/Free Full Text].

52.   Ono, S., J. Guntupalli, and T. D. DuBose, Jr. Role of H+-K+-ATPase in pHi regulation in inner medullary collecting duct cells in culture. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F852-F861, 1996[Abstract/Free Full Text].

53.   Palmer, L. G., and G. Frindt. Regulation of apical membrane Na and K channels in rat renal collecting tubules by aldosterone. Semin. Nephrol. 12: 37-43, 1992[Medline].

54.  Rector, F. C., Jr., H. A. Bloomer, and D. W. Seldin. Effect of potassium deficiency on the reabsorption of bicarbonate in the proximal tubule of the rat kidney. J. Clin. Invest. 43: 1976-1982.

55.   Rosenbaum, B., M. J. Kinney, F. C. Scudder, V. A. DiScala, and R. M. Stein. Effect of potassium depletion on renal tubular sodium and water reabsorption in the dog. Am. J. Physiol. 222: 928-937, 1972[Medline].

56.   Sangan, P., V. M. Rajendran, A. S. Mann, M. Kashgarian, and H. J. Binder. Regulation of colonic H-K ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion. Am. J. Physiol. 272 (Cell Physiol. 41): C685-C696, 1997[Abstract/Free Full Text].

57.   Schwartz, G. J., and M. B. Burg. Mineralocorticoid effects on cation transport by cortical collecting tubules in vitro. Am. J. Physiol. 235 (Renal Fluid Electrolyte Physiol. 4): F576-F585, 1978[Free Full Text].

58.   Seldin, D. W., and F. C. Rector. The generation and maintenance of metabolic alkalosis. Kidney Int. 1: 306-321, 1972[Medline].

59.   Shull, G. E., and J. Lingrel. Molecular cloning of the rat stomach (H+ + K+)-ATPase. J. Biol. Chem. 261: 16788-16791, 1986[Abstract/Free Full Text].

60.   Silver, R. B., H. Choe, and G. Frindt. Low NaCl diet increases H-K ATPase in intercalated cells from rat cortical collecting duct. Am. J. Physiol. 275 (Renal Physiol. 44): F94-F102, 1998[Abstract/Free Full Text].

61.   Silver, R. B., and G. Frindt. Functional identification of H-K-ATPase in intercalated cells of cortical collecting tubule. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F259-F266, 1993[Abstract/Free Full Text].

62.   Silver, R. B., G. Frindt, P. Mennitt, and L. Satlin. Characterization and regulation of H-K ATPase in intercalated cells of rabbit cortical collecting duct. J. Exp. Zool. 279: 443-455, 1997[Medline].

63.   Silver, R. B., P. A. Mennitt, and L. M. Satlin. Stimulation of apical H-K-ATPase in intercalated cells of cortical collecting duct with chronic metabolic acidosis. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F539-F547, 1996[Abstract/Free Full Text].

64.   Soleimani, M., J. A. Bergman, M. A. Hosford, and T. D. McKinney. Potassium depletion increases Na+/H+ exchange and Na+:HCO2-3:HCO-3 cotransport in rat renal cortex. J. Clin. Invest. 86: 1076-1083, 1990[Medline].

65.   Soleimani, M., and G. Singh. Physiologic and molecular aspects of the Na+/H+ exchangers in health and disease processes. J. Investig. Med. 43: 419-430, 1995[Medline].

66.   Stokes, J. B. Potassium secretion by cortical collecting tubule: relation to sodium reabsorption, luminal sodium concentration and transepithelial voltage. Am. J. Physiol. 241 (Renal Fluid Electrolyte Physiol. 10): F395-F402, 1981[Abstract/Free Full Text].

67.   Swarts, H. G. P., C. H. Klaassen, F. M. Schuurmans Stekhoven, and J. H. H. De Pont. Sodium acts as a potassium analog on gastric H,K-ATPase. J. Biol. Chem. 270: 7890-7895, 1995[Abstract/Free Full Text].

68.   Tannen, R. L. Relationship of renal ammonia production and potassium homeostasis. Kidney Int. 11: 453-456, 1977[Medline].

69.   Tomita, K., J. J. Pisano, and M. A. Knepper. Control of sodium and potassium transport in the cortical collecting tubule of the rat. Effects of bradykinin, vasopressin and deoxycorticosterone. J. Clin. Invest. 76: 132-136, 1985[Medline].

70.   Tsuchiya, K., G. Giebisch, and P. A. Welling. Molecular characterization and distribution of H/K-ATPase catalytic subunit gene products in the kidney (Abstract). J. Am. Soc. Nephrol. 4: 881, 1993.

71.   Wall, S. M., P. Mehta, and T. D. Dubose. Chronic dietary K+ depletion increases total and SCH 28080-sensitive bicarbonate absorption in IMCDt (Abstract). J. Am. Soc. Nephrol. 8: 12, 1997.

72.   Wall, S. M., A. V. Truong, and T. D. DuBose. H+-K+-ATPase mediates net acid secretion in rat terminal inner medullary collecting duct. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F1037-F1044, 1996[Abstract/Free Full Text].

73.   Wallmark, B., C. Briving, K. Frykland, R. Munson, J. Mendlein, E. Rabon, and G. Sachs. Inhibition of gastric H+,K+-ATPase and acid secretion by SCH 28080, a substituted pyridyl(1,2a)imidazole. J. Biol. Chem. 262: 2077-2084, 1987[Abstract/Free Full Text].

74.   Wang, T., G. Malnic, G. Giebisch, and Y. L. Chan. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule. J. Clin. Invest. 91: 2776-2784, 1993[Medline].

75.   Wang, Z., N. Baird, H. Shumaker, and M. Soleimani. Potassium depletion and acid-base transporters in rat kidney: differential effect of hypophysectomy. Am. J. Physiol. 272 (Renal Physiol. 41): F736-F743, 1997[Abstract/Free Full Text].

76.   Wang, Z., H. Rabb, T. Craig, C. Burnham, G. Shull, and M. Soleimani. Ischemic-reperfusion injury in the kidney: overexpression of colonic H-K-ATPase and suppression of NHE-3. Kidney Int. 51: 1106-1115, 1997[Medline].

77.   Wang, Z., R. B. Silver, G. Frindt, J. Galla, and M. Soleimani. Renal colonic H+-K+-ATPase (cHKA) upregulation in Na depletion hypotonicity: possible roles in acid-base homeostasis (Abstract). J. Am. Soc. Nephrol. 8: 13, 1997.

78.   Weiner, I. D., and A. E. Milton. H+-K+-ATPase in rabbit cortical collecting duct beta -type intercalated cell. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F518-F530, 1996[Abstract/Free Full Text].

79.   Wingo, C. S. Active proton secretion and potassium absorption in the rabbit outer medullary collecting duct. J. Clin. Invest. 84: 361-365, 1989[Medline].

80.   Wingo, C. S., and F. Armitage. Rubidium absorption and proton secretion by rabbit outer medullary collecting duct via H-K-ATPase. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F849-F857, 1992[Abstract/Free Full Text].

81.   Wingo, C. S., K. M. Madsen, A. Smolka, and C. C. Tisher. H-K-ATPase immunoreactivity in cortical and outer medullary collecting duct. Kidney Int. 38: 985-990, 1990[Medline].

82.   Wingo, C. S., and A. J. Smolka. Function and structure of H-K-ATPase in the kidney. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F1-F16, 1995[Abstract/Free Full Text].

83.   Younes-Ibraham, M., C. Barlet-Bas, B. Buffin-Meyer, L. Cheval, R. Rajerison, and A. Doucet. Ouabain-sensitive and ouabain-insensitive K-ATPases in rat nephron: effect of K depletion. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F1140-F1147, 1995.

84.   Zhou, X., and C. S. Wingo. Mechanisms of rubidium permeation by rabbit cortical collecting duct during potassium restriction. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F1134-F1141, 1992[Abstract/Free Full Text].

85.   Zhou, X., and C. S. Wingo. Stimulation of total CO2 flux by 10% CO2 in rabbit CCD: role of an apical SCH-28080 and Ba-sensitive mechanism. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F114-F120, 1994[Abstract/Free Full Text].


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