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
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ABSTRACT |
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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, HCO3, 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
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INTRODUCTION |
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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.
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THE HKAS: NEPHRON SEGMENT DISTRIBUTION |
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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
-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
-type IC (81).
Another immunocytochemistry study in rat CD demonstrated that HKAg
colocalized with H-ATPase in both
-type and
-type ICs, suggesting
an apical location for HKAg in
-type ICs and a basolateral location
in
-type ICs (7). Perfusion studies on isolated rabbit CCDs have
shown that Sch-28080-sensitive HKA exists in
-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 -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
-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 theOther 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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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 -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
-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).
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HKA: FUNCTIONAL STUDIES |
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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 HCO3 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 -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, theFunctional 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
HCO3 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 -subunit (HKA
2b) has been cloned
(37). The HKA
2b is present in the cortex, outer medulla, and inner
medulla and shows greater upregulation than the original
2-subunit
in K+ depletion (37). The renal
cell distribution of HKA
2b and its regulation in other
pathophysiological states remains to be determined.
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HKA: INHIBITOR SENSITIVITY |
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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.
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HKA: ADAPTIVE REGULATION |
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Regulation of HKA has been studied in several pathophysiological states
including K+ depletion, NaCl
deficiency, acid-base perturbations, and in conditions associated with
increased HCO3 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 HCOMicroperfusion experiments on OMCD from
K+-depleted rabbits have shown
that HKA contributes significantly to
HCO3 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
HCO3 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, HCO3 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. HCO3 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 HCO3 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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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 -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 -type and
-type. In the bicarbonate secreting
-type IC model there is an apical Cl
/HCO
3
exchanger and HKA and a basolateral
H+-ATPase. The
-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
-type or
-type IC. In addition to the
K+ reabsorption via the apical
HKA, in the
-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.
|
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
HCO3 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
HCO3-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 HCO3 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
HCO3 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
HCO3 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 -subunit. The strategy toward this end was to
generate disruption of the
-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
-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,
HCO3 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.
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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).
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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).
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