Division of Nephrology, Hypertension and Transplantation, Gainesville Veterans Affairs Medical Center, Gainesville, Florida 32610-0224
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ABSTRACT |
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Both acidosis and hypokalemia stimulate renal ammoniagenesis, and both regulate urinary proton and potassium excretion. We hypothesized that ammonia might play an important role in this processing by stimulating H+-K+-ATPase-mediated ion transport. Rabbit cortical collecting ducts (CCD) were studied using in vitro microperfusion, bicarbonate reabsorption was measured using microcalorimetry, and intracellular pH (pHi) was measured using the fluorescent, pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Ammonia caused a concentration-dependent increase in net bicarbonate reabsorption that was inhibited by luminal addition of either of the H+-K+-ATPase inhibitors, Sch-28080 or ouabain. The stimulation of net bicarbonate reabsorption was not mediated through apical H+-ATPase, basolateral Na+-K+-ATPase, or luminal electronegativity. Although ammonia caused intracellular acidification, similar changes in pHi induced by inhibiting basolateral Na+/H+ exchange did not alter net bicarbonate reabsorption. We conclude that ammonia regulates CCD proton and potassium transport, at least in part, by stimulating apical H+-K+-ATPase.
proton-potassium-exchanging ATPase; kidney tubules; collecting ducts; hydrogen ion concentration; proton-transporting ATP synthase; rabbits
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INTRODUCTION |
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AMMONIA is a low-molecular-weight molecule that plays an important role in renal physiology. It is produced in the proximal tubule (12, 33, 34), concentrated in the renal interstitium by the loop of Henle (6, 15), and secreted into the luminal fluid in the collecting duct (6, 13, 14, 25). Ammonia is the primary component of net acid excretion under basal conditions, and is the primary mechanism by which the kidney increases net acid excretion in response to metabolic acidosis (27, 60).
Whether ammonia plays a role in acid-base homeostasis beyond its role as a urinary constituent is not clear. However, two common clinical conditions, metabolic acidosis and hypokalemia, are associated with increased ammoniagenesis, loop of Henle-mediated ammonia reabsorption, and interstitial ammonia concentrations (12, 23). Both conditions are also associated with increased urinary proton excretion and with decreased urinary potassium excretion (28, 37). At present, no unifying signaling pathway completely explains the association of both increased renal proton secretion and decreased renal potassium excretion with both metabolic acidosis and hypokalemia.
We hypothesized that ammonia might stimulate proton secretion and potassium reabsorption in the renal cortical collecting duct (CCD), a major site for acid-base transport and for potassium transport. We studied the effect of ammonia on acid-base transport, and we determined whether the stimulation observed was mediated through effects of ammonia on H+-K+-ATPase, H+-ATPase, or both. Since ammonia affects both transepithelial voltage (16) and intracellular pH (pHi) (30, 49, 50), and both may regulate CCD acid-base transport, we examined whether ammonia regulates transport through either of these mechanisms. Our results show that ammonia stimulates CCD net bicarbonate reabsorption by increasing H+-K+-ATPase-mediated proton secretion and that this effect is not due to regulation of basolateral Na+-K+-ATPase, transepithelial voltage, or pHi. Thus ammonia may play an important role as a signaling molecule that regulates CCD proton and potassium transport.
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METHODS |
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Microperfusion. Standard in vitro microperfusion techniques utilizing female New Zealand White rabbits (1.5-2 kg) were used (30, 48-50, 53, 54, 57). The solutions used were artificial solutions and, unless otherwise mentioned, contained (in mM) 119.2 NaCl, 3 KCl, 25 NaHCO3, 2 KH2PO4, 1 sodium acetate, 1.2 CaCl2, 1 MgSO4, 5 alanine, and 8.3 glucose. The solutions were equilibrated with 5% CO2-95% O2 and had osmolality adjusted to 290 ± 7 mosmol/kgH2O with NaCl. NH4Cl substituted for NaCl in both the luminal and peritubular solutions when ammonia was used. Chloride-free solutions substituted gluconate for chloride and increased total calcium to 4.0 mM to account for complexing with gluconate. Unless specified otherwise, the ammonia concentration was 10 mM. Most studies used an ~1.5 ml bath chamber that was thermostatically controlled to 37°C in which the peritubular solution was continuously exchanged at 0.3 ml/min. Some studies measuring pHi used a low-volume, laminar flow bath chamber to which preheated, continuously bubbled solutions were delivered at ~6 ml/min (49, 50, 54). At least 30 min was allowed prior to any measurements and between experimental periods.
Bicarbonate transport. Transepithelial bicarbonate transport
was measured using standard techniques (2, 3, 57, 61). Briefly,
perfused fluid was collected in a calibrated pipette of known volume.
The total CO2 (tCO2) concentration, which is predominantly bicarbonate at physiological pH, was measured using either a picapnotherm (WPI, Sarasota, FL) or a flow-through
ultramicrofluorometer (WPI) (43). Similar results were obtained with
each technique, and were combined for analysis. Net bicarbonate
transport, JtCO2, was calculated using the formula
JtCO2 = Vo × (C1 C2)/L, where Vo is the
collected fluid rate in nanoliters per minute, C1 and C2 are the tCO2 contents in the
perfusate (C1) and collected fluid (C2), and
L is the tubule length in millimeters. The luminal flow rate
was maintained at ~4 nl/min by adjusting the hydrostatic perfusion
pressure. Bicarbonate transport was measured two to four times during
each time period and averaged to yield a single measurement.
Exhaustively dialyzed [3H]inulin was added to the perfusate in all experiments measuring bicarbonate transport, and experiments with leak rates exceeding 5% were discarded. Transepithelial voltage was measured using an agarose electrode connected to the perfusion pipette and referenced against the bathing solution and recorded using a high-impedance electrometer.
Intracellular pH. pHi was measured using standard
techniques (30, 31, 49-53). Briefly, intercalated cells were
loaded with the fluorescent, pH-sensitive dye,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),
by adding the acetoxymethyl ester of BCECF (BCECF-AM), 15 µM, to the
luminal solution for ~5 min (48). An ~15-µm diameter region was
excited at 490 and 440 nm, and emission was measured at 530 nm. The
490/440 ratio was calibrated to pHi at the end of the
experiment using the high-potassium-nigericin technique (48). An A-type
intercalated cell (A cell) was identified as an intercalated cell
without apical
Cl/HCO
3 exchange
activity, and a B-type intercalated cell (B cell) was identified as an
intercalated cell with
Cl
/HCO
3 exchange
activity, as we have previously described (30, 31, 49-53).
Chemicals. BCECF-AM was obtained from Molecular Probes (Eugene, OR). Sch-28080 was the kind gift of Dr. James Kaminski (Schering, Bloomfield, NJ). All other chemicals were from Sigma Chemical (St. Louis, MO).
Statistics. Results are presented as mean ± SE. The data were analyzed using paired Student's t-test and ANOVA, as appropriate, and P < 0.05 was used as evidence of statistical significance.
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RESULTS |
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Net bicarbonate transport. Our first set of studies examined
the effect of ammonia on CCD acid-base transport. Figure
1A summarizes the results of these
studies. Net bicarbonate reabsorption averaged 10.7 ± 3.9 pmol · mm1 · min
1
in the absence of ammonia and 17.7 ± 3.3 pmol · mm
1 · min
1
in the presence of ammonia (P < 0.05 vs. no ammonia,
n = 6). In time control studies, in which a sham change in the
luminal and peritubular solutions was made between the basal and
control period, net bicarbonate reabsorption averaged 8.5 ± 2.6 in
the basal period, and was not significantly different in the control period, 7.4 ± 5.1 pmol · mm
1 · min
1 [P = not significant
(NS) vs. basal, n = 3]. Figure 1B summarizes these results. Thus the increase in net bicarbonate reabsorption seen
in Fig. 1A is due to ammonia and cannot be ascribed to
time-dependent changes. Finally, ammonia caused concentration-dependent
stimulation of net bicarbonate reabsorption (P < 0.02 by
ANOVA). Figure 1C summarizes these results. We conclude that
ammonia causes concentration-dependent stimulation of CCD net
bicarbonate reabsorption.
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Effect of proton pump inhibitors. Ammonia could increase net
bicarbonate reabsorption through effects on apical
H+-K+-ATPase, H+-ATPase, or both.
Because conditions associated with increased intrarenal ammonia
concentrations, such as metabolic acidosis and hypokalemia, are
associated with decreased urinary potassium excretion (28, 37), we
tested whether ammonia might regulate net bicarbonate reabsorption by
stimulating a potassium reabsorbing H+-K+-ATPase. Figure
2 summarizes these results. When apical
H+-K+-ATPase was inhibited with luminal
Sch-28080 (105 M), bicarbonate
reabsorption averaged 12.3 ± 1.9 pmol · mm
1 · min
1
in the absence of ammonia, and was not increased by ammonia, 13.4 ± 2.4 pmol · mm
1 · min
1
(P = NS vs. basal, n = 5). Thus ammonia increases CCD
net bicarbonate reabsorption through effects on a luminal
Sch-28080-sensitive transporter, most likely an apical
H+-K+-ATPase.
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Because some H+-K+-ATPase isoforms are
inhibited by the digitalis glycoside, ouabain (8, 9, 26, 32), the next
experiments determined whether ammonia stimulates a ouabain-sensitive
H+-K+-ATPase. Figure
3A summarizes the results. With
ouabain (103 M) in the luminal solution,
initial bicarbonate reabsorption averaged 14.5 ± 2.4 pmol · mm
1 · min
1
and was not increased, instead it was slightly decreased, by ammonia
addition, 11.6 ± 1.9 pmol · mm
1 · min
1
(P < 0.02 vs. basal, n = 5). Why ammonia inhibited
luminal bicarbonate reabsorption when luminal ouabain was present is
unclear. However, it is unlikely to be due to effects on an apical
Na+-K+-ATPase, as this transporter is not
present at the apical membrane of CCD cells (39). More important, these
studies suggest that ammonia increases net bicarbonate reabsorption
through stimulation of a luminal Sch-28080- and luminal
ouabain-sensitive transporter, most likely an apical
H+-K+-ATPase.
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However, ouabain also inhibits Na+-K+-ATPase,
raising the possibility that the effects of ouabain might be due to
access to the peritubular solution and inhibition of basolateral
Na+-K+-ATPase. To test this possibility, we
added ouabain to the peritubular solution at a concentration
(105 M) that should inhibit CCD
Na+-K+-ATPase (11), but not
H+-K+-ATPase (8, 26, 32, 59), and determined
whether this would inhibit the effects of ammonia on net bicarbonate
reabsorption. Figure 3B summarizes these experiments. With
peritubular ouabain (10
5 M) present,
bicarbonate reabsorption averaged 5.6 ± 1.6 pmol · mm
1 · min
1
in the absence of ammonia and was significantly stimulated by ammonia,
11.9 ± 2.8 pmol · mm
1 · min
1
(P < 0.01 vs. absence of ammonia, n = 5). Inhibiting
basolateral Na+-K+-ATPase does not alter the
increase in net bicarbonate reabsorption caused by ammonia (P = NS by ANOVA). We conclude that ammonia stimulates net bicarbonate
reabsorption through effects that are independent of basolateral
Na+-K+-ATPase and are sensitive to luminal ouabain.
H+-ATPase is the primary CCD proton transporter stimulated
by in vitro metabolic acidosis (45) and is the primary apical proton
transporter that regulates pHi in both the A cell and the B
cell, the cells responsible for CCD acid-base transport (30, 51). The
next protocol tested whether ammonia stimulates net proton secretion by
activating H+-ATPase. Figure 4
summarizes the results. In the presence of luminal bafilomycin
A1 (108 M), a potent
H+-ATPase inhibitor (55), net bicarbonate reabsorption
averaged 6.5 ± 2.0 pmol · mm
1 · min
1
and was significantly increased by ammonia to 13.3 ± 2.8 pmol · mm
1 · min
1
(P < 0.05 vs. basal, n = 6). Luminal bafilomycin
A1 did not alter ammonia's stimulation of bicarbonate
transport (P = NS by ANOVA). Thus ammonia stimulates
bicarbonate reabsorption by stimulating a luminal Sch-28080- and
ouabain-sensitive H+-K+-ATPase and not by
stimulating a bafilomycin A1-sensitive
H+-ATPase.
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Effect of ammonia on transepithelial voltage. Active sodium
transport can regulate bicarbonate transport through changes in luminal
electronegativity. However, this is unlikely to be the mechanism
through which ammonia regulates CCD bicarbonate transport. In the
current studies ammonia did not change CCD transepithelial voltage
significantly (6.0 ± 2.2 mV, basal;
4.7 ± 1.9 mV, with ammonia; P = NS, n = 5). Also, as reported
above, inhibiting active sodium reabsorption with peritubular ouabain
did not prevent ammonia from increasing net bicarbonate reabsorption
(see Fig. 3B).
Effect of ammonia on pHi. Because ammonia can alter pHi and because acute pHi changes are believed to regulate proton secretion, we next tested whether ammonia stimulated bicarbonate transport through changes in pHi. To do so, we used a three-part experiment. First, we determined the effect of prolonged ammonia incubation on pHi. Because CCD net bicarbonate transport is believed to be mediated by the intercalated cells, of which there are two major subtypes, the A cell and the B cell, and not by principal cells, we measured the effect of prolonged ammonia incubation on A cell and B cell pHi. Then, we identified that inhibiting basolateral Na+/H+ exchange activity would cause similar changes in steady-state A cell and B cell pHi as ammonia. Finally, we tested whether acidifying the A cell and B cell by inhibiting basolateral Na+/H+ exchange would cause changes in CCD bicarbonate transport similar to those caused by ammonia.
The first step was to determine the effect of prolonged ammonia
incubation on pHi. The addition of ammonia acutely
alkalinized both the A cell and B cell (not shown), followed shortly
thereafter by intracellular acid loading. As can be seen in Fig.
5, ammonia decreased A cell and B cell
pHi below initial pHi within as little as 10 min. The acidification was maximal at 30 min, decreasing A cell
pHi from 7.48 ± 0.06 to 7.27 ± 0.05 (P < 0.001, n = 12) and B cell pHi from 7.43 ± 0.03 to
7.11 ± 0.03 (P < 0.001, n = 14) and did not
increase any further at 60 min (pHi =
0.05 ± 0.03 and
0.02 ± 0.02 vs. pHi at 30 min,
P = NS and P = NS, n = 4 and n = 4, for
the A cell and B cell, respectively). These changes were ammonia
dependent; both A cell and B cell pHi were unchanged in the
absence of ammonia (data not shown). Thus, in contrast to ammonia's
immediate effect to cause intracellular alkalinization, prolonged
incubations can cause net intracellular acidification.
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Second, we identified that inhibiting basolateral
Na+/H+ exchange would cause similar changes in
A cell and B cell pHi as did ammonia incubation. As Fig.
6A shows, adding the Na+/H+ exchange
inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 106 M, to the peritubular solution
decreased significantly both A cell and B cell pHi
(
pHi =
0.17 ± 0.07 and
0.23 ± 0.07, P < 0.05 and P < 0.025, respectively). Inhibiting
basolateral Na+/H+ exchange activity with
peritubular EIPA acidified the A cell and the B cell to the same extent
as ammonia.
Third, we tested whether acidifying cells with EIPA would have the same
effect on CCD bicarbonate transport as did ammonia. These results are
summarized in Fig. 6B. Under basal
conditions, CCD reabsorbed bicarbonate at a rate averaging 4.7 ± 1.0 pmol · mm1 · min
1
(n = 5). Peritubular EIPA addition
(10
6 M) did not alter net bicarbonate
reabsorption significantly, 4.2 ± 1.1 pmol · mm
1 · min
1
(P = NS, n = 5). Thus intracellular acidification is
unlikely to be the primary mechanism through which ammonia increases
H+-K+-ATPase-dependent bicarbonate
reabsorption.
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DISCUSSION |
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The current study identifies several new and important features regarding the role of ammonia in ion transport. First, ammonia causes concentration-dependent stimulation of CCD bicarbonate reabsorption through activation of H+-K+-ATPase-dependent proton secretion. Second, ammonia causes intracellular acidification, but this is not sufficient to explain how ammonia increases net bicarbonate reabsorption. Finally, these findings suggest that ammonia can function as an extracellular signaling molecule that regulates CCD proton and potassium transport.
The first major finding of the current study is that ammonia increases rabbit CCD net bicarbonate reabsorption. The range of ammonia concentrations chosen in this study parallel those present in the late distal tubule (23, 56) and thus are likely to be similar to those to which the CCD is exposed. The findings in this study may be applicable to both the cortical and medullary collecting duct. For example, ammonia increases bicarbonate reabsorption in both the rat CCD and inner medullary collecting duct (IMCD) (25, 46). Furthermore, differences between the effects of 4 and 10 mM ammonia were also seen in the inner stripe of the outer medullary collecting duct (14). Thus the current study is both consistent with and, more important, extends these previous studies in several ways.
First, ammonia specifically activates H+-K+-ATPase-mediated proton secretion. In the current study, two H+-K+-ATPase inhibitors, Sch-28080 and ouabain, each completely inhibited the effect of ammonia on net bicarbonate reabsorption, whereas the H+-ATPase inhibitor, bafilomycin A1, had no significant effect. Thus ammonia's effect on proton secretion does not reflect a general effect on apical proton transporters. Stimulation of an H+-K+-ATPase is consistent with the observation that ammonia increases CCD 86Rb+ (a surrogate for K+) reabsorption (16). The lack of stimulation of an electrogenic H+-ATPase is consistent with the observation that ammonia does not alter transepithelial voltage independent of its effects on sodium reabsorption (16). An alternative, but unlikely, possibility is that NH+4 serves as substrate for apical K+/NH+4 exchange. Because NH+4 is a weak acid, active NH+4 secretion could mediate the increased rate of net bicarbonate reabsorption. However, ammonia secretion is mediated by NH3 diffusion, not by NH+4 secretion (17, 24), making apical K+/NH+4 exchange unlikely to mediate a significant proportion of apical NH+4 secretion, and thus bicarbonate reabsorption. Instead, ammonia appears to increase net bicarbonate reabsorption by stimulating electroneutral H+-K+-ATPase-mediated proton secretion and potassium reabsorption. Whether ammonia also inhibits CCD unidirectional bicarbonate secretion cannot be determined definitively at present. However, our preliminary studies suggest that ammonia, in addition to its effects on proton secretion and luminal bicarbonate reabsorption, may also inhibit B cell unidirectional bicarbonate secretion (47). Identifying all of ammonia's cellular effects in the CCD will be an important field for future study.
Which H+-K+-ATPase isoform ammonia activates
cannot be determined definitively at present. The -subunit of the
H+-K+-ATPase
-
heterodimer mediates
catalytic and transport activity, and it determines the inhibitor
sensitivity of the holoenzyme (58, 59). Of the known renal
H+-K+-ATPase
-isoforms, HK
1
is Sch-28080 sensitive and ouabain insensitive (58, 59), whereas
HK
2a and HK
2b are Sch-28080 insensitive and ouabain sensitive when expressed in heterologous
expression systems (8, 26, 32). A renal HK
2c was
recently cloned (7); its sensitivity to Sch-28080 and ouabain remains
to be determined. Thus the pharmacological characterization of the
ammonia-stimulated H+-K+-ATPase differs from
the characteristics of HK
1, HK
2a, and
HK
2b. Whether these differences are due to unidentified
regulatory molecules or whether ammonia stimulates a novel
H+-K+-ATPase
-isoform will be an important
field for further study.
Although H+-K+-ATPase is the primary proton transporter mediating the response to ammonia, it is not the primary CCD proton transporter in all conditions. Apical H+-K+-ATPase did not significantly contribute to basal bicarbonate reabsorption in either the current study or another study (61). Similarly, it mediates a relatively small role in both A cell and B cell pHi regulation, at least compared with H+-ATPase (30, 51). Finally, different conditions that activate CCD bicarbonate reabsorption appear to activate different apical proton transporters. For example, in vitro metabolic acidosis primarily activates bicarbonate reabsorption through activation of H+-ATPase (45), whereas in vitro respiratory acidosis increases bicarbonate reabsorption by activating H+-K+-ATPase, without apparent effects on H+-ATPase (61). Understanding the cellular signaling pathways through which these different stimuli increase CCD bicarbonate reabsorption through differing proton transporters will be an important issue for future study.
Ammonia appears to stimulate CCD net bicarbonate reabsorption through effects that are unrelated to its effects on principal cell-mediated electrogenic sodium transport and transepithelial voltage. In the current study, inhibiting active sodium reabsorption with peritubular ouabain did not prevent ammonia from increasing net bicarbonate reabsorption, nor did ammonia significantly alter transepithelial voltage. In another study, peritubular ouabain did not alter rat CCD net bicarbonate reabsorption when ammonia was present (24). Another report found that ammonia inhibited CCD sodium reabsorption and decreased luminal electronegativity (16), effects which would be expected to decrease bicarbonate reabsorption and thus cannot mediate the stimulation seen in the current study. Why the current study and the study by Hamm et al. (16) found different effects of ammonia on luminal electronegativity is unclear. Also, why there was a tendency for basal bicarbonate reabsorption to be decreased in the presence of peritubular ouabain is unclear, but this may be related to inhibition of intercalated cell basolateral Na+-K+-ATPase (39), with secondary effects on membrane potential or on intracellular sodium, potassium, or calcium. More important, multiple studies confirm that ammonia's effects on bicarbonate transport are not due to its effects on principal cell-mediated sodium transport.
Another important finding is that ammonia can acidify CCD intercalated cells. Although acute ammonia exposure caused acute intracellular alkalinization, as seen in previous studies (30, 49, 51), this was followed by sustained acid loading that caused net intracellular acidification. Although ammonia acidified the B cell slightly more than the A cell, the functional significance of this difference is not clear, as ammonia's effects on bicarbonate transport appear unrelated to its effects on pHi. The ability of ammonia to cause intracellular acidification is not unique to CCD intercalated cells. Similar results have been observed in central nervous system (CNS) neurons (5, 35), and intracellular acidification may mediate ammonia's inhibition of CCD sodium reabsorption (16). Why basolateral Na+/H+ exchange (30, 49) did not mediate any apparent pHi recovery is not clear, but this may be related to possible substitution of NH+4 for H+ on Na+/H+ exchange (22) or inhibition of Na+/H+ exchange by ammonia (42).
Ammonia could induce intracellular acidification through any of several possible mechanisms. Because ammonia exists in solution predominantly as NH+4, a weak acid, it is possible that NH+4 uptake causes the intracellular acidification. This is consistent with observations in the medullary thick ascending limb of the loop of Henle (21). Alternatively, ammonia might directly regulate proton or bicarbonate transporters. For example, ammonia increases AE2 activity independent of its effects on pHi (19). Further studies will be necessary to differentiate between these possibilities.
Although ammonia acidified CCD intercalated cells, changes in pHi may not be the primary mechanism through which ammonia, or other stimuli, regulate CCD bicarbonate transport. For example, metabolic acidosis (decreased extracellular bicarbonate) increases CCD net bicarbonate reabsorption (4) by stimulating H+-ATPase (45), whereas respiratory acidosis (increased extracellular PCO2) has been reported either to not alter bicarbonate reabsorption (4) or to increase net bicarbonate reabsorption by stimulating H+-K+-ATPase (61). Despite these differing effects on net bicarbonate reabsorption, metabolic acidosis and respiratory acidosis cause similar changes in pHi (49, 50). Similarly, isoproterenol acidifies the B cell and did not alter A cell pHi (18) yet decreases net bicarbonate reabsorption (41). Since there appears to be a dissociation between effects of experimental stimuli on pHi and on net bicarbonate reabsorption, it appears unlikely that pHi is the most immediate signal through which these stimuli, and, in particular, ammonia, regulate proton secretion and bicarbonate reabsorption. However, we cannot exclude the possibility that pHi may have a more minor effect on CCD bicarbonate transport.
A final consideration is that ammonia may function as a signaling molecule that can regulate ion transport. Several studies have shown that the proximal tubule produces ammonia and the rate of production is physiologically regulated by at least two stimuli, metabolic acidosis and hypokalemia (23, 33), that also regulate CCD ion transport. The current study shows that ammonia causes concentration-dependent stimulation of CCD bicarbonate transport. Ammonia also inhibits CCD sodium reabsorption and potassium secretion (16), and it increases IMCD bicarbonate reabsorption (46). In other organs, ammonia inhibits colonic chloride secretion (38), and it regulates CNS astrocyte glutamine uptake (10), taurine release (1), and calcium uptake (36). What might be the physiological benefit of ammonia-mediated stimulation of CCD H+-K+-ATPase transport? One major stimulus that increases renal ammoniagenesis and cortical ammonia level is metabolic acidosis (56). It is possible that the increased ammoniagenesis and renal ammonia content signal increased collecting duct bicarbonate reabsorption and luminal acidification. Hypokalemia is a second stimulus that increases ammoniagenesis. In hypokalemia, an important role of the increased ammoniagenesis may be to stimulate CCD H+-K+-ATPase-mediated potassium reabsorption. Supporting this hypothesis is that increasing ammoniagenesis, by infusing its metabolic precursor glutamine, decreases urinary potassium excretion (40, 44) by increasing potassium reabsorption at a site distal to the micropuncturable late distal tubule (20). Both the current study and a study by Hamm et al. (16) demonstrating that ammonia increases CCD 86Rb+ reabsorption, a measure of active potassium reabsorption, localize at least some of this effect to the CCD. Thus ammonia is produced by the proximal tubule and regulates the transport activities of collecting duct cells, and conditions that stimulate its production are associated with changes in ion transport that ammonia can mediate. Ammonia-mediated signaling may play an important role in both acid-base and potassium homeostasis.
An unexpected observation in these studies was that CCD reabsorbed luminal bicarbonate under basal conditions. Most previous studies have shown that CCD exhibit little to no net bicarbonate transport under basal conditions (4, 29, 45), although some have shown bicarbonate reabsorptive rates similar to the current study (61). Why basal bicarbonate transport was different from reported in some other studies is unclear. However, it was unlikely to be due to metabolic acidosis, a known stimulus of CCD bicarbonate reabsorption (29), as urinary pH in animals treated similarly as the ones used in the current study averaged ~8.3 (unpublished results). Similarly, the animals were on a normal potassium content rabbit diet, making induction of H+-K+-ATPase by potassium deficiency unlikely.
In conclusion, the current study demonstrates several important new observations. First, ammonia stimulates proton secretion through effects on H+-K+-ATPase. Second, ammonia causes intracellular acidification, but this does not appear to be the primary mechanism through which ammonia stimulates proton secretion. Finally, ammonia may function as an extracellular signaling molecule regulating CCD proton and potassium transport. These observations may have important implications for our understanding of renal acid-base and potassium homeostasis.
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ACKNOWLEDGEMENTS |
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We thank Jeannette Lynch for technical assistance and Gina Cowsert for secretarial assistance. We also thank Dr. C. Craig Tisher and Dr. Steven L. Gluck for continued support
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45788 (to I. D. Weiner) and DK-49750 (to C. S. Wingo) and by Department of Veterans Affairs Merit Review Grants (to I. D. Weiner and C. S. Wingo).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. D. Weiner, Division of Nephrology, Hypertension and Transplantation, Univ. of Florida, P. O. Box 100224, Gainesville, FL 32610-0224 (E-mail: WeineID{at}ufl.edu).
Received 23 April 1999; accepted in final form 17 August 1999.
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