1 Center for Nephrology, Departments of Medicine and Physiology, University College London Medical School, London W1N 8AA, United Kingdom; and 2 Department of Nephrology, Faculty of Medicine, Second University of Naples, Naples 803131, Italy
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ABSTRACT |
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We have studied
the effects of acute respiratory alkalosis (ARALK, hyperventilation)
and acidosis (ARA, 8% CO2),
chronic respiratory acidosis (CRA; 10%
CO2 for 7-10 days), and
subsequent recovery from CRA breathing air on loop of Henle (LOH) net
bicarbonate flux
(JHCO3)
by in vivo tubule microperfusion in anesthetized rats. In ARALK blood,
pH increased to 7.6, and blood bicarbonate concentration
([]) decreased from 29 to 22 mM. Fractional urinary bicarbonate excretion
(FEHCO3)
increased threefold, but LOH
JHCO3
was unchanged. In ARA, blood pH fell to 7.2, and blood
[
] rose from 28 to 34 mM; FEHCO3 was
reduced to <0.1%, but LOH
JHCO3 was unaltered. In CRA, blood pH fell to 7.2, and blood
[
] increased to >50
mM, whereas
FEHCO3
decreased to <0.1%.
JHCO3 was reduced by ~30%. Bicarbonaturia occurred when CRA rats breathed air, yet LOH
JHCO3
increased (by 30%) to normal. These results suggest that LOH
JHCO3
is affected by the blood-to-tubule lumen
[
] gradient and
backflux. When the usual
perfusing solution at 20 nl/min was made
free, mean
JHCO3
was
34.5 ± 4.4 pmol/min compared with 210 ± 28.1 pmol/min plus
. When a low-NaCl
perfusate (to minimize net fluid absorption) containing mannitol and
acetazolamide (2 × 10
4 M, to abolish
H+-dependent
JHCO3)
was used,
JHCO3
was
112.8 ± 5.6 pmol/min. Comparable values for
JHCO3
at 10 nl/min were
35.9 ± 5.8 and
72.5 ± 8.8 pmol/min, respectively. These data indicate significant backflux of
along the LOH, which depends on
the blood-to-lumen [
] gradient; in addition to any underlying changes in active acid-base transport mechanisms,
permeability and backflux are important determinants of LOH
JHCO3
in vivo.
loop of Henle; bicarbonate transport; respiratory acidosis; respiratory alkalosis; permeability; backflux
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INTRODUCTION |
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THE KIDNEY PLAYS an important role in the homeostatic response to acute and chronic acid-base disturbances by changes in reabsorption of filtered bicarbonate in exchange for secreted H+ and in the excretion of nonbicarbonate buffers such as phosphate and ammonium. Several groups have shown that the proximal tubule is the major site of bicarbonate reabsorption (2, 3, 30) and that net transport adjusts to systemic acid-base changes (13, 14, 28, 31). However, more distal nephron segments may also participate in the renal adaptation to acid-base disturbances (26). Among the distal nephron segments, the loop of Henle (LOH) reabsorbs ~10-15% of the filtered bicarbonate (6, 15). We have shown previously that bicarbonate transport in this segment in vivo is not saturated under normal acid-base conditions and is mediated by luminal Na+/H+ exchange and H+-adenosinetriphosphatase (H+-ATPase) (8).
The LOH is composed of several subsegments, including the thick ascending limb (TAL). Good (21) has shown that the perfused TAL in vitro reabsorbs bicarbonate and is responsible for a significant portion of total LOH bicarbonate transport. He has also reported that bicarbonate reabsorption in the TAL is stimulated by metabolic acidosis and inhibited by metabolic alkalosis (22). We have demonstrated similar effects of metabolic acid-base disturbances along the LOH in vivo (9). However, the effect of respiratory acid-base changes on the LOH has not been investigated. In contrast to metabolic acid-base disturbances, in respiratory abnormalities, the blood bicarbonate concentration decreases in alkalosis and increases in acidosis. In low-resistance leaky epithelia, like the proximal tubule and part of the LOH, the blood-to-tubule lumen bicarbonate concentration gradient is probably also an important determinant of net bicarbonate absorption in vivo.
To investigate the response of the LOH under conditions of altered respiratory acid-base conditions and fixed-end proximal delivery of bicarbonate, we have done a tubule microperfusion study on the effect of acute and chronic respiratory acid-base manipulations on bicarbonate reabsorption along the rat LOH in vivo. Our data show that acute respiratory alkalosis and acidosis have no effect on LOH bicarbonate transport, whereas, in chronic respiratory acidosis, bicarbonate absorption is depressed but rises during partial recovery when blood bicarbonate concentration falls as urinary bicarbonate excretion increases. The microperfusion data can be explained in terms of changes in bicarbonate backflux (from blood to tubule fluid) as a consequence of alterations in the blood-to-lumen bicarbonate concentration gradient. The results of additional microperfusion experiments designed to minimize the active transport component of bicarbonate absorption and to assess the magnitude of bicarbonate backflux along the LOH are consistent with this conclusion.
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METHODS |
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Preparation of animals. We did experiments on a total of 45 male Sprague-Dawley rats (220 ± 4 g body wt) kept in cages at 21°C in controlled daylight and fed until the day of the study. They were anesthetized intraperitoneally with Inactin (Promonta), using a dose of 120 mg/kg body wt, tracheostomized, placed in the right lateral position on a thermoregulated table (37°C), and prepared for micropuncture in the usual way. In brief, the right carotid artery was catheterized to record blood pressure and take blood for measurements of hematocrit, arterial pH, and blood gases. The left jugular vein was cannulated with PE-50 tubing and used for intravenous infusion via a syringe pump (Razel, Semat Technical) of a Ringer-saline solution (125 mM NaCl + 25 mM NaHCO3) at 4 ml/h. The left kidney was exposed through a flank incision, made free of perirenal fat, and immobilized in a Lucite chamber with 3% agar in 0.9% saline. The kidney was bathed with prewarmed (37°C) paraffin oil. The bladder and left ureter were catheterized with PE-90 and PE-10 tubing, respectively, for collection of urine.
Tubule
microperfusion. We performed
continuous microperfusion of superficial LOHs in vivo to measure
bicarbonate and fluid transport under conditions of fixed flow rate and
bicarbonate delivery. Briefly, a perfusion pipette was inserted into
the last surface loop of proximal tubule, and a castor oil block was
placed upstream of the perfusion pipette. Microperfusion was started at
20 nl/min with a thermally shielded microperfusion pump (Hampel, Frankfurt, Germany). The perfusion solution contained (in mM) 128 NaCl,
13 NaHCO3, 3.8 KCl, 1 MgCl2, 0.38 NaH2PO4,
and 1.62 Na2HPO4
(280 mosmol/kgH2O). FD & C blue
dye (0.07%) and 12.5 µCi/ml [14C]inulin were added to the
perfusion solution. Net fluid flux
(Jv) and net
bicarbonate transport rate
(JHCO3) were measured under the following conditions:
1) acute respiratory alkalosis
induced by mechanical hyperventilation [SAR 830 mechanical ventilator, CWE; tidal volume
(VT) = 10.5 ml, respiratory
rate = 75-80 breaths/min],
2) acute respiratory acidosis
induced by mechanical ventilation with 8%
CO2
(VT = 7.5, respiratory rate = 40 breaths/min), and 3) chronic
respiratory acidosis (CRA) produced by maintaining animals in a 10%
CO2 atmosphere for 7-10 days
and subsequently during recovery when breathing air. To maintain the acidotic state during micropuncture, the rats continued to breath 10%
CO2 through a purpose-designed,
positive-pressure hood delivering a constant 10%
CO2-90% air mixture. This hood
was removed during the "recovery" phase. In acute respiratory
acidosis, to assist mechanical ventilation and to maintain normoxia and
hemodynamic stability, it was necessary to use the neuromuscular
blocking agent gallamine triethiodide (Rhone-Poulenc Rorer; 5 mg/kg iv bolus followed by 15 mg · kg1 · h
1
iv infusion) in addition to Inactin anaesthesia. Tubule microperfusion did not begin until at least 45 min after surgery, when the animal was
equilibrated and stable and at least 30 min after the subsequent acute
changes in mechanical ventilation and respiratory acid-base status.
To estimate whole-loop bicarbonate permeability, we did separate microperfusion experiments in normal rats with a modified solution previously reported by McKay and Peterson (32) to prevent net fluid and chloride reabsorption along the LOH, perfusing at 10 and 20 nl/min. This was bicarbonate free and contained (in mM) 93 NaCl, 5 KCl, 4.1 MgSO4, 1 NaH2PO4, 1 CaCl2, 4 urea, 16 sodium gluconate, 71 mannitol, 0.2 acetazolamide, FD & C blue dye (0.05%), and 12.5 µCi/ml [14C]inulin (300 mosmol/kgH2O).
Except for the CRA group, each treatment group served as its own control. Transport data are given per individual loop, since it has been shown (43) that the cortical LOH is a nephron segment of essentially constant length (~6-7 mm).
Whole kidney clearance. In the same groups of animals, glomerular filtration rate (GFR) and sodium, potassium, and bicarbonate excretion were measured in the micropunctured kidney. After a priming dose of 20 µCi [3H]inulin in 0.5 ml 0.9% saline, the maintenance intravenous Ringer-bicarbonate solution delivered [3H]inulin at 20 µCi/h. After a 45-min equilibration, the first of three to five 30 to 45 min urine collections began. Carotid artery blood samples were taken at the start and end of each experiment and at ~1 h after any change in acid-base status.
Analytical methods. Tubule fluid total CO2 concentration was measured by microcalorimetry (Picapnotherm, World Precision Instruments). To avoid loss of CO2, all mineral oil used (to bathe kidney surface, to collect tubule fluid samples, and to cover samples for measurement) was equilibrated to cortical carbon dioxide tension (PCO2) values (16) with a solution containing 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer and 48 mM NaHCO3 and equilibrated with 6.7% of CO2. Each sample analysis was bracketed by running standards of known NaHCO3 concentration. The blood acid-base status of each animal and calculated blood bicarbonate concentration were measured with a blood-gas analyzer (Corning model 178). [14C]inulin radioactivity was measured by a liquid scintillation counter (Parkard Tri-Carb 2000CA). Plasma Na+ concentration [Na+] and K+ concentration ([K+]) were measured by ion-sensitive microelectrodes (Corning model 614).
Calculations and statistical analysis. Single-kidney GFR and fractional electrolyte excretion were calculated using standard clearance formulas.
In the micropuncture experiments, the perfusion rate achieved in vivo
was obtained from the rate of fluid collected from the early distal
tubule, multiplied by the ratio of inulin concentrations in collected
and perfused fluids. The perfusion pump was calibrated by
timed collections of perfusion fluid delivered directly into counting
vials for measurements of [14C]inulin concentrations; calculated
Jv (in nl/min)
was the difference between perfusion and collection rates.
JHCO3
(in pmol/min) was calculated from the amount of bicarbonate delivered
in the perfusion pipette minus the amount collected in the collection
pipette, according to the following formulas:
Vp = (Inc/Inp) × (Vc),
where Vp is the
calculated perfusion rate, Inc and
Inp are the collected and perfused
inulin counts, respectively, and
Vc is the
collection rate;
Jv = Vp Vc; and
JHCO3 = (Cp × Vp)
(Cc × Vc), where
Cp and
Cc are the bicarbonate
concentrations of the perfused and collected fluids.
A sample collection was considered acceptable if Vp was within 15% of the calibrated microperfusion pump rate.
Statistical analysis was by one-way analysis of variance (ANOVA). For JHCO3, because it is load dependent, one-way analysis of covariance (ANCOVA) was used with bicarbonate load as the covariate (8). ANOVA and ANCOVA were followed by comparison with the appropriate control (post hoc if P < 0.05) using the least significant difference test. A value of P < 0.05 was considered to be statistically significant. Data are given as means ± SE.
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RESULTS |
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Blood gases and electrolytes. Table
1 summarizes the arterial blood composition
data of the animals studied in each treatment group. Values of pH,
PCO2, and blood
concentration
([
]) were as expected
for each respiratory acid-base disturbance. Mechanical ventilation on
air was associated with a decrease in plasma
[K+]. In acute
respiratory alkalosis, the rise in blood pH was associated with a fall
in PCO2, blood
[
], and
plasma [K+]. In acute
respiratory acidosis, a low blood pH was associated with a high
PCO2 and the formation of new
bicarbonate with an increased blood
[
] but no
significant change in plasma
[K+]. In CRA, blood pH
was reduced, PCO2 was high, and blood [
] was
significantly elevated; plasma
[K+] was also
significantly increased, but, during partial recovery (breathing air),
it fell.
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Renal clearance data. Table
2 shows the effect of each respiratory
acid-base manipulation on GFR and urinary excretion of Na+,
K+, and
HCO. In the same group of rats,
mechanical ventilation per se had a small effect on GFR, reducing it
from 0.72 ± 0.10 (data not shown in Table 2) to 0.56 ± 0.07 ml · min
1 · 100 g
1
(P > 0.05). Acute respiratory
alkalosis did not significantly change GFR, sodium fractional excretion
(FENa), or potassium
fractional excretion (FEK), whereas
FEHCO3
increased threefold. In acute respiratory acidosis, GFR did not change,
but FENa and
FEHCO3 were
reduced. In CRA, GFR fell slightly,
FENa and FEK remained unchanged, and
FEHCO3 was low.
During the partial recovery phase, CRA (when breathing
air) was characterized by a marked increase in bicarbonate excretion
(~30-fold), a modest rise in potassium excretion, but no change in
GFR.
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LOH microperfusion data. Table 3 and Fig. 1 show the microperfusion data obtained in the three different respiratory acid-base states. In the same group of animals, mechanical ventilation on air had no significant effect on JHCO3 (data not shown in Table 3) compared with spontaneous respiration of air (211.8 ± 20.7 vs. 210.5 ± 12.2 pmol/min, P > 0.05). In acute respiratory alkalosis, JHCO3 did not change (182.1 ± 12.7 vs. 200.6 ± 11.7 pmol/min, P > 0.05). In contrast, FEHCO3 increased significantly in the same experimental animals from 0.45 ± 0.14 to 1.39 ± 0.34% (P < 0.05, see Table 2). In acute respiratory acidosis, JHCO3 did not change (210.5 ± 12.2 vs. 189.8 ± 13.6 pmol/min, P > 0.05), although the final urinary excretion of bicarbonate was significantly decreased (Table 2). An unexpected finding was that LOH bicarbonate reabsorption was significantly depressed in CRA animals compared with control animals (143.1 ± 9.7 vs. 182.1 ± 12.7 pmol/min, P < 0.05), whereas FEHCO3 was low. When these chronically CO2-adapted animals were switched to breathing air, urinary bicarbonate excretion increased, and JHCO3 rose significantly from 143.1 ± 9.7 to 174.6 ± 6.3 pmol/min (P < 0.05). Only small and barely significant changes in fluid reabsorption were observed in each treatment group.
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Table 4 summarizes the results of further
microperfusion experiments designed to assess the contribution of
bicarbonate backflux along the LOH to net bicarbonate reabsorption.
Experiments were done at 20 nl/min using the standard perfusion
solution with and without bicarbonate (perfusates
A and B,
respectively). In the absence of bicarbonate in the perfusion solution,
there was an influx of bicarbonate of 34.5 ± 4.4 pmol/min
into the collected fluid compared with a net efflux of bicarbonate of
210.0 ± 28.1 pmol/min (in the presence of ~15 mM bicarbonate in
the perfusate). When experiments were done at two different perfusion
rates of 20 and 10 nl/min using a solution containing bicarbonate but
designed to minimize net fluid reabsorption (perfusate
C), net bicarbonate reabsorption was greater at the
higher perfusion rate, but fractional reabsorption of bicarbonate was
similar at the two perfusion rates. This is consistent with our
previously published observations on load and flow dependence of
bicarbonate absorption along the LOH (8). However, when compared with
the standard perfusion solution containing bicarbonate
(perfusate A) perfused at 20 nl/min, fractional bicarbonate reabsorption was reduced, indicating that a
component of net bicarbonate absorption depends on net fluid reabsorption (solvent drag). When this perfusion solution
(perfusate C) was bicarbonate free
(perfusate D), a substantial influx
of bicarbonate into the collected fluid occurred at both 20 and 10 nl/min,
47.4 ± 7.8 and
35.9 ± 5.8 pmol/min,
respectively. In addition, when acetazolamide was added
(perfusate E) to abolish carbonic
anhydrase-dependent H+ secretion,
a significantly higher influx of bicarbonate into the collected fluid
was observed at both perfusion rates,
112.8 ± 5.6 and
72.5 ± 8.8 pmol/min, respectively.
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DISCUSSION |
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In acute respiratory alkalosis and acidosis, there is a rapid (within minutes) change in blood bicarbonate concentration because of blood buffering. Unlike in metabolic acid-base disturbances, in respiratory acid-base imbalance, blood pH and blood bicarbonate concentration change in opposite directions. In respiratory alkalosis, pH increases and blood bicarbonate concentration decreases as PCO2 falls; in respiratory acidosis, pH decreases and blood bicarbonate concentration increases as PCO2 rises. In respiratory alkalosis and acidosis, various methods in different tissues have shown that intracellular pH parallels changes in extracellular pH, although the magnitude of change in pH may be different (1, 5, 24, 41). The changes in plasma potassium concentration within each treatment group, including mechanical ventilation per se, are not easily explained (Table 1). Although metabolic acidosis and alkalosis are associated with hyperkalemia and hypokalemia, respectively, the pattern in respiratory acid-base disturbances is variable and clearly depends on more than changes in systemic pH and intracellular H+ buffering. Alterations in peripheral sympathetic nerve activity and relative changes in plasma concentrations of norepinephrine and epinephrine seem to be important, although species dependent (27, 39), and may also influence net urinary bicarbonate excretion (33) (Table 2).
Because the rate of H+ secretion
by cells is dependent on the intracellular
H+ concentration, the two main
acid-extruding mechanisms,
Na+/H+
exchange and H+-ATPase, should be
suppressed in respiratory alkalosis and stimulated in respiratory
acidosis. There is extensive literature reporting increased activity of
both
Na+/H+
exchange and H+-ATPase in
respiratory acidosis in different epithelia, including the kidney.
Proximal tubule membrane vesicle studies and in vitro tubule
microperfusion experiments have demonstrated stimulation of apical
Na+/H+
exchange and basolateral
Na+-
cotransport activity in respiratory acidosis (25, 40, 45, 46). In the
collecting duct and toad bladder, an increase in
PCO2 has been shown to stimulate
H+-ATPase activity (36, 38). This
has also been confirmed in the collecting duct by immunocytochemical
studies (4). Assays of renal
H+-ATPase and
H+-K+-ATPase
activities following respiratory acid-base changes show them to be
increased in respiratory acidosis after 24 h and decreased in
respiratory alkalosis after 6 h (17). There are no reports of the
effect of more acute hypocapnea on
H+-ATPase activity or
Na+/H+
exchange.
When tubular bicarbonate load is fixed and is independent of GFR, renal
tubular absorption of bicarbonate depends on luminal H+ secretion (34); an increase in
H+ secretion will enhance and a
decrease will depress net bicarbonate reabsorption. Therefore, we would
expect increased bicarbonate reabsorption in respiratory acidosis and
decreased reabsorption in respiratory alkalosis. However, another
important variable to consider is the peritubular capillary blood
bicarbonate concentration and the transepithelial bicarbonate
concentration gradient. This could influence net bicarbonate
reabsorption by affecting tubule cell bicarbonate exit
(Na+-
cotransport) and paracellular bicarbonate flux. In metabolic acid-base
disturbances, these changes are in parallel and therefore additive: in
metabolic acidosis, increased H+
secretion is associated with a reduced blood bicarbonate concentration and reduced bicarbonate backflux (from blood to lumen); in metabolic alkalosis, decreased H+ secretion
is associated with a raised blood bicarbonate concentration and
increased bicarbonate backflux. The situation is different in
respiratory acid-base disorders, in which changes in
PCO2 parallel tubule cell
H+ secretion, and blood
bicarbonate concentration also mirrors
PCO2, i.e., increased blood
PCO2 and bicarbonate in acidosis and
decreased blood PCO2 and bicarbonate
in alkalosis. Thus an increase in peritubular capillary blood
bicarbonate concentration may oppose and a decrease in blood
bicarbonate concentration favor net bicarbonate reabsorption. Such an
effect is likely to be more important in leaky epithelia, like the
proximal tubule, than in tight epithelia, like the distal nephron.
Because the LOH is an intermediate segment of mixed epithelia, the
influence of changes in blood bicarbonate concentration on net
bicarbonate transport is less clear.
We observed no change in net bicarbonate absorption in the LOH in either acute respiratory alkalosis or acidosis. In the proximal tubule, earlier in vitro microperfusion and in vivo micropuncture studies reported inhibition of net bicarbonate absorption in alkalosis and slight stimulation in respiratory acidosis (13, 23). However, Ullrich et al. (42) and Zeidel and Seifter (46) found no change in proximal tubule acidification in respiratory acidosis. A more recent in vivo study by Santella et al. (35) also found no effect under these conditions when changes in the bicarbonate filtered load were taken into account. These authors concluded that the major determinant of net bicarbonate transport in this part of the nephron is the bicarbonate load. Along the LOH, we have also shown that bicarbonate reabsorption is load dependent (8). To control for this, we did our microperfusion experiments using a fixed load of bicarbonate. Therefore, any changes in net bicarbonate transport along the LOH must reflect alterations in the activity of the acid-base transporters and the degree of bicarbonate backflux. The two main luminal acidifying mechanisms along the LOH are the Na+/H+ exchanger and the H+-ATPase (8). As already mentioned, these transporters are stimulated by a rise in PCO2, and they are probably inhibited by a fall in PCO2. Because we found no change in JHCO3 in acute respiratory alkalosis and acidosis, this suggests that any change in active transport activity is countered by an opposite change in bicarbonate backflux, which in turn depends on the blood-to-lumen bicarbonate concentration gradient, i.e., an increased gradient in respiratory acidosis and a decreased gradient in respiratory alkalosis. This interpretation is strengthened by our observation that, in chronic respiratory acidosis, in which there was a steeper blood-to-lumen bicarbonate gradient than in acute respiratory acidosis, there was inhibition of net bicarbonate transport. In addition, on switching these acidotic chronically CO2-adapted animals from 10% CO2 to air, there was a rapid increase in JHCO3 to control values (see Fig. 1). Although the bloodto-lumen bicarbonate concentration gradient decreased and therefore the amount of bicarbonate backflux, the blood bicarbonate concentration was still significantly elevated. In chronic respiratory acidosis, it has been shown that stimulated Na+/H+ exchanger activity remains increased for up to 24 h after withdrawal of the elevated CO2 (45). Thus it is possible that some of the rebound in JHCO3 that we observed (Fig. 1) is also due to a persistent enhancement of Na+/H+ exchanger activity in the presence of a smaller blood-to-lumen bicarbonate gradient.
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The renal clearance data are similar to earlier studies showing an
initial increase in bicarbonate excretion in acute respiratory alkalosis and a decrease in acute and chronic respiratory acidosis (37). However, a striking observation was the marked increase in
urinary bicarbonate excretion following the switch from breathing high
CO2 to air in chronically
CO2-adapted animals. This cannot be explained by a change in GFR or bicarbonate reabsorption along the
proximal nephron, including the LOH, but must be due to a rapid
inhibition of bicarbonate absorption beyond the LOH. It seems likely
that this is mediated by a change in
H+-ATPase, which is known to be
regulated by PCO2 and blood bicarbonate concentration (17). It has been shown that luminal membrane
insertion and withdrawal of this transporter are fast and sensitive to
changes in PCO2 (36). In addition, there might also be a relative increase in apical
Cl/
exchange activity and bicarbonate secretion.
To confirm the importance of bicarbonate backflux in the LOH, we did further experiments to assess whole segment transepithelial permeability to bicarbonate. Although these experiments do not discriminate among any differences in bicarbonate permeability of the various nephron segments that make up the LOH, they do take account of the corticomedullary solute gradients in vivo. Bicarbonate permeability has been estimated in more homogeneous nephron segments of the proximal and distal tubules in vivo, and the results show a decrease in permeability from proximal to distal nephron (11, 12, 20, 29, 44). As illustrated in Fig. 2, which shows only the mean and directional changes in net bicarbonate flux, our observations under conditions of minimal net fluid reabsorption and inhibition of active H+ secretion along the LOH in vivo demonstrate that passive bicarbonate backflux can be up to 50% of active bicarbonate reabsorption. In keeping with these findings is the observation of Byers at al. (7), who perfused the LOH in vivo with a bicarbonate-free solution and found an accumulation of 4 mM bicarbonate in the collected fluid, a value almost identical with our own (see Table 4). Moreover, in our previous study of LOH perfusion in which there was a large blood-to-lumen bicarbonate gradient, we found that inhibition of luminal Na+/H+ exchange and H+-ATPase changed net bicarbonate flux from reabsorption to secretion (10). Under normal acid-base conditions in vivo, a corticomedullary gradient for interstitial bicarbonate concentration (measured as total CO2) exists and favors net bicarbonate transfer from vasa recta to juxtamedullary LOHs (18), which could facilitate adaptation to an alkali load (19).
In conclusion, our results show that, when the LOH is perfused in vivo with a fixed bicarbonate load, changes in respiratory acid-base balance do not alter net bicarbonate reabsorption. This apparent lack of effect of respiratory alkalosis or acidosis on LOH bicarbonate reabsorption could be explained by concomitant changes in the blood-to-lumen bicarbonate concentration gradient offsetting any inhibition or stimulation of H+ secretion induced by a fall or rise in PCO2, respectively. In chronic respiratory acidosis, there was indirect evidence for an underlying stimulation of LOH acidification that was opposed by the associated rise in blood bicarbonate concentration and blood-to-lumen bicarbonate gradient. Finally, the importance of bicarbonate backflux along the LOH was confirmed by additional experiments, the results of which demonstrate that in vivo bicarbonate permeability and passive flux of bicarbonate are major determinants of net bicarbonate transport, and this is not always considered under in vitro experimental conditions.
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ACKNOWLEDGEMENTS |
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G. Capasso and R. Unwin both thank the British Council, Ministero dell'Universitá della Ricecca Scientifica e Technologica (MURST), and the North Atlantic Treaty Organization for collaborative awards. R. Unwin also thanks the Wellcome Trust and the St. Peter's Trust for support.
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FOOTNOTES |
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G. Capasso was supported by a grant from MURST (60%).
Address for reprint requests: R. Unwin, Center for Nephrology, Depts. of Medicine and Physiology, University College London Medical School, Institute of Urology and Nephrology, Middlesex Hospital, Mortimer Street, London W1N 8AA, UK.
Received 18 September 1996; accepted in final form 19 June 1997.
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