Endothelin-1 increases rat distal tubule acidification in vivo
Donald E.
Wesson and
George M.
Dolson
Departments of Internal Medicine and Physiology, Texas Tech
University Health Sciences Center, Lubbock 79430; and Department of
Internal Medicine, Veterans Affairs Medical Center, Baylor College
of Medicine, Houston, Texas 77030
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ABSTRACT |
Because endothelin
receptor inhibition blunts increased distal tubule acidification
induced by dietary acid, we examined whether endothelin-1 (ET-1)
increases acidification of in vivo perfused distal tubules of
anesthetized rats. ET-1 was infused intra-aortically (1.4 pmol · kg
1 · min
1)
into control animals and into those with increased distal tubule HCO3 secretion induced by drinking
80 mM NaHCO3 solution for
7-10 days. ET-1 increased distal tubule acidification in both
control and NaHCO3 animals.
Increased acidification in control animals was mediated by increased
distal tubule H+ secretion (23.7 ± 2.2 vs. 18.7 ± 1.7 pmol · mm
1 · min
1,
P < 0.05) with no changes in
HCO3 secretion. By contrast, ET-1 increased distal tubule acidification in
NaHCO3 animals predominantly by
decreasing HCO3 secretion
(
9.5 ± 1.0 vs.
18.7 ± 1.8 pmol · mm
1 · min
1,
P < 0.001) with less influence on
H+ secretion. When indomethacin
was infused (83 µg · kg
1 · min
1)
to inhibit synthesis of prostacyclin, an agent previously shown to
increase HCO3 secretion in the
distal tubule, ET-1 increased distal tubule
H+ secretion in both control (24.3 ± 2.2 vs. 15.7 ± 1.6 pmol · mm
1 · min
1,
P < 0.02) and
NaHCO3 (20.0 ± 2.0 vs. 13.6 ± 1.4 pmol · mm
1 · min
1,
P < 0.05) without affecting
HCO3 secretion. The data show that ET-1 increases distal tubule acidification in vivo and can do so by
increasing H+ secretion and by
decreasing HCO3 secretion when the
latter is augmented by dietary
NaHCO3.
acid; bicarbonate; indomethacin; micropuncture; prostacyclin
 |
INTRODUCTION |
DIETARY ACID-BASE CHANGES alter distal tubule
acidification in a way that helps to maintain normal acid-base
homeostasis, but the diet-induced factors that modulate acidification
in this nephron segment are not clear. Close association of distal
convoluted and connecting tubules with renal microvasculature in the
cortical labyrinth (12) provides opportunity for modulation of distal tubule transport by renal vascular endothelium through paracrine communication. Recent studies are consistent with such paracrine communication mediated by prostacyclin (31), an agent synthesized by
renal microvascular endothelium (18). Dietary
HCO3 increases urine prostacyclin
excretion (31), and inhibition of prostacyclin synthesis blunts the
decrease in distal tubule acidification induced by dietary
HCO3 (31). Furthermore, systemic
prostacyclin infusion decreases distal tubule acidification in both
HCO3-ingesting and control animals
(31). By contrast, dietary acid ingested as (NH4)2SO4
increases endothelin-1 (ET-1) addition to renal interstitial fluid and
pharmacological inhibition of endothelin receptors in acid-ingesting
animals blunts increased distal tubule acidification induced by this
dietary maneuver (32). The latter studies are consistent with
endothelin-stimulated distal tubule acidification mediated either
directly or indirectly by endothelin-inhibiting actions of agents that
decrease acidification in this nephron segment.
The present studies tested the hypothesis that ET-1 directly increases
distal tubule acidification. We measured components of distal tubule
acidification in control and
NaHCO3-ingesting (latter with
reduced distal tubule acidification) rats systemically infused with
ET-1. We further tested whether ET-1 effects on distal tubule
acidification were evident when prostacyclin synthesis was inhibited.
The data show that ET-1 increases distal tubule acidification in both
control and HCO3-ingesting animals
in the absence and presence of inhibited prostaglandin synthesis.
Increased acidification was mediated predominantly by augmented
H+ secretion in control animals
and predominantly by reduced HCO3 secretion in NaHCO3-ingesting
animals with intact prostacyclin synthesis. The data support the
hypothesis that ET-1 directly increases distal tubule acidification.
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MATERIALS AND METHODS |
The present studies tested the hypothesis that systemically
administered ET-1 increased distal tubule acidification. The studies were done in 250- to 295-g male and female Munich-Wistar rats (Harlan
Sprague Dawley, Houston, TX) fed minimum electrolyte diet (ICN
Nutritional Biochemicals, Cleveland, OH) and distilled
H2O. Because we hypothesized that
ET-1 increases distal tubule acidification, we also studied animals
that ate the same diet but drank 80 mM NaHCO3 drinking solution 7-10
days prior to study, a protocol that reduces distal tubule
acidification (30). Animals were infused intra-aortically with ET-1
(1.4 pmol · kg
1 · min
1)
beginning with the 1-h equilibration period and continuing throughout the subsequent 1-h micropuncture period. This ET-1 dose in rats does
not change mean blood pressure, glomerular filtration rate, or renal
blood flow (9). Because dietary
HCO3 increases urine prostacyclin
synthesis (31) and prostacyclin decreases distal tubule acidification
(31), we explored whether ET-1 effects on acidification were evident
when prostacyclin synthesis was inhibited. Prostacyclin synthesis was
inhibited with intravenous indomethacin infused at 83 µg · kg
1 · min
1,
a dose that inhibits prostacyclin synthesis in rats (19).
Micropuncture protocol. Animals were
prepared for micropuncture of accessible distal tubules as described
(33). This distal nephron segment is composed of multiple epithelia (4)
but will hereafter be referred to as "distal tubule" for
simplicity. Mineral oil blocks were placed in a proximal and distal
loop of surface distal tubules, which were perfused at the early distal
flow rate measured in situ (6 nl/min) (29), calibrated in vitro, and
verified in vivo (33). Transepithelial potential difference was
measured after perfusate collection for each solution (33). An injected latex cast determined perfused tubule length after subsequent acid
digestion of the kidney (33). Anerobically obtained arterial (0.35 ml)
and stellate vessel blood plasma (33) was analyzed for total
CO2
(tCO2) using flow-through
fluorometry (22) and for pH, PCO2,
and electrolytes using standard techniques (29). Diet but not drinking
solution was withheld the evening before study, yielding higher
baseline HCO3 reabsorption (14) and permitting differences in HCO3
reabsorption to be more clearly seen.
Table 1 depicts perfusate composition.
Standard perfusate HCO3
concentration ([HCO3])
and Cl
concentration
([Cl
]) were 5 and 40 mM, respectively, to approximate
their concentrations in control early distal tubule fluid (29).
Perfusate 1 contained HCO3 for measurement of net
HCO3 reabsorption and for
calculation of luminal H+
secretion as previously described (33). Perfusate
2 was HCO3 free to
assess blood-to-lumen HCO3
accumulation and to calculate a linear flux coefficient for
HCO3 transport into the distal
tubule lumen, as done previously (33) and described below.
Perfusate 3 was identical to
2, except that it also contained 0.5 mM acetazolamide. Acetazolamide inhibits
HCO3 (33) and
H+ secretion (1) in the in vivo
perfused rat distal tubule. Thus measuring luminal
HCO3 accumulation and voltage when
perfusing with a zero-HCO3,
zero-Cl
, and
acetazolamide-containing solution allows calculation of passive
blood-to-lumen transepithelial
HCO3 permeability, as done previously in our laboratory (33) and by others (2).
Perfusate 3 served this purpose.
Solutions 4 and
5 were identical to
1 and 2, respectively, except that the
former solutions were Cl
free. Solutions 4 and
5 permitted determination of
HCO3 transport parameters when
tubule HCO3 transport was
inhibited by reduced luminal
Cl
(13). All perfusates
contained raffinose to minimize fluid transport and permit more focused
study of HCO3 transport. Three selected distal tubules were perfused in each of four animal groups with and without systemic indomethacin infusion:
H2O ingesting (control),
H2O + ET-1,
NaHCO3 ingesting, and
NaHCO3 + ET-1. In one set of
animals, an identified distal tubule was perfused with solution 1, another with
solution 2, and the third with
solution 3. In another animal set, one
distal tubule was perfused with solution
4 and another with solution
5. Perfusate order was random.
Analytical methods. After experiment
termination, initial and collected perfusate and stellate vessel plasma
were analyzed for inulin (29) and for
tCO2 using flow-through
ultrafluorometry (22) as done previously (30). All tubule fluid and
plasma [HCO3] were
measured on the experimental day by comparing fluorescence of a 7- to
8-nl aliquot (corrected for that of a distilled
H2O blank run with each sample
group) to a standard curve. A standard curve was constructed for each
sample group using an identical volume of the following
NaHCO3 standards: 0, 2.5, 5, 10, 25, and 50 mM. The best-fit linear regression for all 20 sample runs
had slope of 83.1 (range, 72.0-88.9) peak units/mM,
y-intercept = 22.1 (range,
16.1-25.9) peak units,
r2 = 0.98, and
regression standard error = 8.9.
Calculations. Net
HCO3 transport was the difference
between the perfused and collected rates. Net
HCO3 reabsorption refers to net
HCO3 transport when perfusing with
initially HCO3-containing solutions. Luminal HCO3
accumulation refers to net HCO3
transport when perfusing with initially
HCO3-free solutions. A positive HCO3 transport indicates net
HCO3 movement out of the lumen
(reabsorption), and a negative value indicates net
HCO3 movement into the lumen (secretion). A linear flux coefficient for
HCO3 transport into the distal
tubule lumen (solutions 2 and
5) or passive
HCO3 permeability (solution 3) was calculated as done
previously (2, 33). HCO3 secretion
was estimated when perfusing with
HCO3-containing solutions by
calculating HCO3 transport into
the lumen using the linear flux coefficient for
HCO3 transport into the distal
tubule lumen derived from perfusing with the
HCO3-free solution (33).
H+ secretion was equated to
absolute HCO3 reabsorption and was
estimated during perfusion with the
HCO3-containing perfusates by
subtracting calculated HCO3
secretion from measured net HCO3
reabsorption as done previously (33). This
H+ secretion quantifying method
assumes that all HCO3 transport from the lumen (absolute HCO3
reabsorption) is mediated by luminal H+ secretion (1). Fluid
reabsorption was the difference between the perfused and collected flow
rates. All transport values were corrected for perfused tubule length
(in mm) determined from latex injection.
Statistical analysis. The data for
each perturbation of individual tubules (1-2
perfusions · each
perturbation
1 · animal
1)
were meaned to obtain a unique value for each animal, and those unique
values for each animal of a group were meaned to obtain a group
average. The Bonferroni method was used for
t-test comparison of means
(P < 0.05) when multiple different
comparisons of the same parameter were done between control and
NaHCO3-ingesting animals.
 |
RESULTS |
Animal growth in response to dietary
protocols. There were no differences in body weight
between H2O- (control) and
NaHCO3-ingesting (NaHCO3) animals at the start
(248 ± 6 and 246 ± 6 g, respectively) or at the end (262 ± 7 and 259 ± 6 g, respectively) of the 1-wk NaHCO3-ingesting or
H2O-ingesting periods. Food intake
was the same between periods and among
NaHCO3 and control groups.
Hemodyanmic response to ET-1. Mean
blood pressure was not different among ET-1-infused
compared with baseline control [114 ± 3 vs. 112 ± 3 mmHg,
respectively; P = not significant
(NS)] or NaHCO3 (106 ± 2 vs. 107 ± 3 mmHg, respectively; P = NS) animals.
Micropuncture data. Plasma electrolyte
and acid-base composition including arterial and stellate vessel plasma
[HCO3] were not
different among groups (data not shown). Table
2 shows that, when pefusing with
solution 1 (5 mM
HCO3), distal tubule net
HCO3 reabsorption was higher in
ET-1-infused compared with the respective baseline group of
NaHCO3 but not control animals. In
subsequent studies, we investigated whether the ET-1-induced increase
in net HCO3 reabsorption in
NaHCO3 animals was meditated by
increased H+ secretion
and/or decreased HCO3
secretion. The method for calculating H+ and
HCO3 secretion when perfusing with
the HCO3-containing solutions requires combining parameters derived from perfusing distal tubules with HCO3-free and
HCO3-containing solutions (see
MATERIALS AND METHODS).
Solution 2 (HCO3 free) served this purpose,
and the perfusion data are in Table 3.
Luminal HCO3 accumulation and linear flux coefficient for HCO3
were significantly lower in the ET-1-infused compared with the
respective baseline group of
NaHCO3 but not control animals.
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Table 3.
Net blood-to-lumen HCO3 flux and permeability in distal
tubules animals perfused with solution 2 (0 HCO3, 40 mM Cl )
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The decrease in luminal HCO3
accumulation induced by ET-1 in
NaHCO3 animals might be due to
decreased cellular HCO3 secretion or to decreased passive transepithelial
HCO3 permeability. To help
distinguish these possibilities, distal tubules were perfused with
solution 3 (zero
HCO3, zero
Cl
, acetazolamide
containing) to calculate passive transepithelial HCO3 permeability as described in
MATERIALS AND METHODS. This parameter
was not different between NaHCO3
or control animals infused with ET-1 compared with those that were not
as shown in Table 4. These data show that
ET-1 did not alter passive HCO3 permeability, supporting that ET-1 increases net
HCO3 reabsorption of
NaHCO3-ingesting animals by
decreasing cellular HCO3
secretion.
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Table 4.
Net blood-to-lumen HCO3 fluxes and permeabilities in distal
tubules of animals perfused with solution 5 (0 HCO3 and
0 Cl with 0.5 mM acetazolamide)
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Perfusion data from solutions 1 and
2 were combined to calculate
H+ and
HCO3 secretion when perfusing with
HCO3-containing solutions as
described in MATERIALS AND METHODS.
Figure 1 shows that calculated HCO3 secretion was lower in
ET-1-infused compared with baseline NaHCO3 animals (
9.8 ± 1.0 vs.
18.7 ± 1.8 pmol · mm
1 · min
1,
respectively; P < 0.01). By
contrast, calculated HCO3
secretion in the ET-1-infused control animals was not different from
the respective baseline group (
3.9 ± 0.5 vs.
5.3 ± 0.6 pmol · mm
1 · min
1,
respectively; P = NS). Figure
2 shows that calculated
H+ secretion in distal tubules of
ET-1-infused compared with baseline was not different in
NaHCO3 animals (21.8 ± 2.0 vs.
22.4 ± 2.3 pmol · mm
1 · min
1,
respectively; P = NS) but was higher
in control animals (24.7 ± 2.3 vs. 17.8 ± 1.7 pmol · mm
1 · min
1,
respectively; P < 0.05). Thus higher
net HCO3 reabsorption induced by
ET-1 in NaHCO3-ingesting animals
was predominately due to decreased HCO3 secretion. In addition, ET-1
increased calculated H+ secretion
in control but not NaHCO3 animals
perfused with solution 1.

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Fig. 1.
HCO3 secretion in distal tubules
perfused with 5 mM HCO3 containing
Cl
(solution 1). Bar labels denote
animal groups; legend denotes those without ( ) and with (+)
endothelin-1 (ET-1). * P < 0.05 vs. respective group without ET-1,
P < 0.05 vs. respective
control.
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Fig. 2.
H+ secretion in distal tubules
perfused with 5 mM HCO3 containing
Cl
(solution 1).
* P < 0.05 vs. respective
group without ET-1.
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Because HCO3 secretion into the
distal tubule lumen decreases net
HCO3 reabsorption and might
influence luminal H+ secretion,
the next series of microperfusion experiments were done with solutions
that were initially Cl
free
to inhibit luminal HCO3 secretion
(13). Table 5 shows that distal tubule net
HCO3 reabsorption was higher in
ET-1-infused compared with the respective baseline group of both
control and NaHCO3-ingesting
animals when each was perfused with solution 4 (5 mM HCO3,
Cl
free). To determine
whether the ET-1-induced increase in net HCO3 reabsorption of control and
NaHCO3 animals was mediated by decreased HCO3 secretion
and/or increased H+
secretion, distal tubules were subsequently perfused with
solution 5 (HCO3 and
Cl
free). Table
6 shows that luminal
HCO3 accumulation and linear flux
coefficient for HCO3 were
significantly lower in the ET-1-infused compared with the respective
baseline group of NaHCO3 but not control animals. The data from perfusions done with
solutions 4 and
5 were combined to calculate
H+ and
HCO3 secretion when perfusing with
the HCO3-containing
solution 4, as described earlier for
perfusions with solutions 1 and
2. Figure
3 shows that calculated
HCO3 secretion was lower in the ET-1-infused compared with baseline
NaHCO3 animals (
5.5 ± 0.6 vs.
9.5 ± 0.8 pmol · mm
1 · min
1,
respectively; P < 0.03).
By contrast, calculated HCO3
secretion in the ET-1-infused control animals was not different from
the respective baseline group (
4.0 ± 0.4 vs.
4.7 ± 0.5 pmol · mm
1 · min
1,
respectively; P = NS). Figure
4 shows that calculated
H+ secretion in ET-1-infused
compared with baseline groups was not different in
NaHCO3 animals (21.7 ± 2.0 vs.
16.8 ± 1.6 pmol · mm
1 · min
1,
respectively; P = NS) but was higher
in control animals (25.0 ± 2.1 vs. 17.5 ± 1.5 pmol · mm
1 · min
1,
respectively; P < 0.05). Thus, when
HCO3 secretion was inhibited with
Cl
-free solutions, the
ET-induced increase in net HCO3
reabsorption in NaHCO3-ingesting
animals was more clearly mediated by decreased HCO3 secretion than by increased
H+ secretion. By contrast, the
ET-1-induced increase in net HCO3 reabsorption of control animals in this setting was due to increased H+ secretion.
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Table 6.
Net blood-to-lumen HCO3 flux and permeability in distal
tubules of animals perfused with solution 5 (0 HCO3,
0 Cl )
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Fig. 3.
HCO3 secretion in distal tubules
perfused with 5 mM HCO3 without
Cl
(solution 4).
* P < 0.05 vs. respective
group without ET-1, P < 0.05 vs. respective control.
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Fig. 4.
H+ secretion in distal tubules
perfused with 5 mM HCO3 without
Cl
(solution 4).
* P < 0.05 vs. respective
group without ET-1.
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Prostacyclin increases distal tubule
HCO3 secretion, and indomethacin
blunts augmented distal tubule
HCO3 secretion induced by dietary
NaHCO3 (31). The following studies
investigated ET-1 effects on distal tubule acidification when
prostacyclin synthesis was inhibited with indomethacin. Table
7 shows that ET-1 increased net HCO3 reabsorption in distal
tubules of both control and
NaHCO3-ingesting animals infused
with indomethacin regardless of whether tubules were perfused with
Cl
-containing
(1) or
Cl
-free solutions
(4). To determine whether the higher
net HCO3 reabsorption of control
and NaHCO3-ingesting animals was
mediated by decreased HCO3
secretion and/or increased
H+ secretion, distal tubules were
subsequently perfused in paired fashion with
HCO3-free solutions that were
either Cl
free
(5) or contained
Cl
(2). Table
8 shows no difference in luminal
HCO3 accumulation and linear flux
coefficient for HCO3 in
ET-1-infused compared with the respective baseline group of control and
NaHCO3 animals. Figures
5 and 7 show that ET-1 had no effect on
HCO3 secretion in distal tubules
of either control or
NaHCO3-ingesting animals infused
with indomethacin. By contrast, Fig. 6
shows that when distal tubules were perfused with
Cl
-containing solutions,
H+ secretion was higher in
ET-1-infused compared with baseline in control (24.3 ± 2.2 vs. 15.7 ± 1.6 pmol · mm
1 · min
1,
P < 0.02) and
NaHCO3 (20.0 ± 1.9 vs. 13.6 ± 1.4 pmol · mm
1 · min
1,
P < 0.05) animals. Similarly, Fig.
8
shows that when distal tubules were perfused with
Cl
-free solutions,
H+ secretion was higher in
ET-1-infused compared with baseline in control (26.5 ± 2.2 vs. 17.6 ± 1.6 pmol · mm
1 · min
1,
P < 0.02) and
NaHCO3 (21.9 ± 2.0 vs. 15.5 ± 1.5 pmol · mm
1 · min
1,
P < 0.05) animals. The data show
that increased H+ secretion
mediates the augmented distal tubule net
HCO3 reabsorption induced by ET-1
in the presence of indomethacin.
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Table 7.
Bicarbonate reabsorption by distal tubules of
NaHCO3-ingesting and control animals perfused with
solutions 1 and 4 (5 mM HCO3, without and with 40 mM
Cl , respectively) and infused with indomethacin
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Table 8.
Net blood-to-lumen HCO3 flux and permeability in distal
tubules of NaHCO3-ingesting and control animals
perfused with solutions 2 and 5 (0 HCO3, without and with
40 mM Cl , respectively) and infused with
indomethacin
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Fig. 5.
HCO3 secretion in distal tubules
perfused with 5 mM HCO3 containing
Cl
(solution 1) in animals infused with
indomethacin.
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Fig. 6.
H+ secretion in distal tubules
perfused with 5 mM HCO3 containing
Cl
(solution 4) in animals infused with
indomethacin. * P < 0.05 vs. respective group without ET-1.
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Fig. 7.
HCO3 secretion in distal tubules
perfused with 5 mM HCO3 without
Cl
(solution 1) in animals infused with
indomethacin.
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Fig. 8.
H+ secretion in distal tubules
perfused with 5 mM HCO3 without
Cl
(solution 4) in animals infused with
indomethacin. * P < 0.05 vs.
respective group without ET-1.
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 |
DISCUSSION |
The factors that mediate changes in distal tubule acidification in
response to altered dietary acid-base content are not clear. The close
association of distal convoluted and connecting tubules with renal
microvasculature in the cortical labyrinth provides opportunity for
modulation of distal tubule transport by renal vascular endothelium
through paracrine communication. Dietary HCO3 increases renal excretion of
prostacyclin, an agent synthesized by renal vascular endothelium (18)
that increases distal tubule HCO3
secretion (31). Thus prostacyclin might mediate such paracrine communication in response to dietary
HCO3 (31). On the other hand,
dietary acid increases renal secretion of ET-1 (32), an agent also
synthesized by renal microvascular endothelium (34). Pharmacological
inhibition of endothelin receptors in acid-ingesting animals blunts the
increase in distal tubule acidification induced by this dietary
maneuver (32). Similarly, endothelins might be paracrine modulators of
distal tubule acidification. This is supported by the presence of
receptors to both ET-1 (26) and prostaglandin
I2 (8) in rat
cortical collecting tubule. ET-1 might increase distal tubule
acidification directly given that it stimulates
Na+/H+
exchange in renal epithelia (3, 5). Alternatively, endothelins might
increase distal tubule acidification indirectly by inhibiting actions
of other agents such as prostacyclin. The present studies tested the
hypothesis that ET-1 directly increases distal tubule acidification.
The data support the hypothesis by showing that ET-1 increases distal
tubule acidification in control and
NaHCO3-ingesting animals, both
with and without inhibited prostacyclin synthesis. These and previous
(32) studies support ET-1 modulation of distal tubule acidification in
vivo.
Endothelins were initially distinguished by their vasoactive effects
(17), but it is now clear that they also modulate epithelial transport.
These agents inhibit the amiloride-sensitive
Na+ channel (15), NaCl
reabsorption (25), and antidiuretic hormone-mediated H2O reabsorption (24) in
collecting tubules. ET-1 also stimulates the
Na+/H+
exchanger (NHE) in renal cortical membrane vesicles (5) and the NHE-3
isoform in renal epithelial cells (3), supporting an endothelin role in
modulating renal acidification in vivo. Na+/H+
exchange sensitive to amiloride analogs mediates a portion of rat
distal tubule H+ secretion
examined in vivo (27) and might be a target of endothelin action.
Dietary acid increases ET-1 addition to renal interstitial fluid and
inhibition of endothelin receptors blunts the augmented distal tubule
acidification induced by this dietary maneuver (32). The latter studies
show that endogenous endothelins mediate increased distal tubule
acidification induced by dietary acid. The present studies show that
ET-1 increases distal tubule acidification directly and not necessarily
by modulating actions of other agents such as prostacyclin. In contrast
to ET-1-stimulated distal tubule acidification, ET-1 inhibited net
HCO3 reabsorption in proximal straight tubules (7). In vitro data suggest that ET-1 stimulates acidification in proximal convoluted tubules (5) consistent with the
directional change in acidification observed for distal tubules in the
present studies. Together, these data suggest that ET-1 has distinct
effects on acidification among nephron segments, but the physiological
meanings of these differences are not known.
The present studies show that ET-1 increases distal tubule
acidification in control animals by increasing
H+ secretion. This was most
clearly shown in distal tubules perfused with
Cl
-free perfusates that
inhibit HCO3 secretion in this
nephron segment (13). ET-1-stimulated
H+ secretion might be due to
augmented
Na+/H+
exchange (3) shown to mediate distal tubule
H+ secretion (27) and/or
possibly to stimulated H+-ATPase
and/or
H+-K+
ATPase activity present in rat cortical collecting tubules (20). By
contrast, ET-1 increased acidification in the
NaHCO3 animals predominantly by
decreasing distal tubule HCO3
secretion. ET-1-stimulated H+
secretion could not be demonstrated in distal tubules of
NaHCO3 animals perfused with
either Cl
-containing or
Cl
-free solutions without
concomitant indomethacin infusion. By contrast, an ET-1-stimulated
distal tubule H+ secretion could
be demonstrated in NaHCO3 animals
concomitantly infused with indomethacin. Although prostacyclin did not
inhibit H+ secretion and
indomethacin did not increase H+
secretion in distal tubules (31), increased renal prostacyclin levels
(or other indomethacin-sensitive agents) induced by dietary NaHCO3 (31) might inhibit the
increase in distal tubule H+
secretion stimulated by ET-1. Further studies are necessary to clarify
this issue.
The cellular signaling mechanism(s) that mediate increased distal
tubule acidification induced by ET-1 was not determined in the present
studies. It was also not clear whether the increased H+ secretion and decreased
HCO3 secretion induced by ET-1
were mediated by similar or separate signaling mechanisms. An
intracellular second messenger that might link ET-1 effects on both
components of distal tubule net
HCO3 reabsorption is adenosine
3',5'-cyclic monophosphate (cAMP). ET-1 inhibits
agonist-stimulated increases in cellular cAMP levels in renal
epithelium (24), a cellular phenomenon that is associated with
augmented HCO3 secretion in cortical collecting tubules (6) and with inhibited NHE activity in
renal brush-border membranes (28). Thus the ET-1-induced decrease in
HCO3 secretion and increase in
H+ secretion in distal tubules of
the present studies might be mediated through reduced and/or
blunted increases in cellular cAMP levels. On the other hand, ET-1 also
stimulates Ca2+ release from
internal stores and its entry into cortical collecting duct cells (11)
consistent with activation of phospholipase C (21). Activation of
phospholipase C has been associated with stimulated NHE activity (16).
Furthermore, activation of B-type endothelin receptors generates nitric
oxide through a tyrosine kinase-dependent and
Ca2+/calmodulin-dependent pathway
(26) and nitric oxide increases rat proximal tubule net
HCO3 reabsorption in vivo (35).
Thus ET-1 might alter
HCO3/H+
secretion by these and possibly other mechanisms.
In summary, the present studies show ET-1 increases distal tubule
acidification in the presence and absence of indomethacin-sensitive products including prostacyclin, consistent with a direct effect of
this agent. The ET-1-induced increased distal tubule acidification is
mediated by stimulated H+
secretion in control animals and primarily by decreased
HCO3 secretion in
NaHCO3-ingesting animals whose
baseline distal tubule HCO3
secretion is higher than control. These data and those of previous
studies (32) support a role for ET-1 in modulating distal nephron
acidification in vivo.
 |
ACKNOWLEDGEMENTS |
We are grateful to Geraldine Tasby and Cathy Hudson for expert
technical assistance, to Edward McGuire for expert animal care, and to
Neil A. Kurtzman for continued intellectual support.
 |
FOOTNOTES |
This work was supported by funds from the Merit Review Program of the
Department of Veterans Affairs, from National Institute of Diabetes and
Digestive and Kidney Diseases Grant 5-RO1-DK-36199-10 [to N. A. Kurtzman (principal investigator)], and by funds from the
Texas Tech University Health Sciences Center.
Address for reprint requests: D. E. Wesson, Texas Tech Univ. Health
Sciences Center, Renal Section, 3601 Fourth St., Lubbock, TX 79430.
Received 9 September 1996; accepted in final form 17 June
1997.
 |
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