1 Instituto de Investigaciones Cardiológicas-Consejo Nacional de Investigaciones Científicas y Técnicas, 1122 Buenos Aires; and 2 Escuela de Ciencia y Tecnología, Universidad Nacional de General San Martín, 1650 San Martín, Argentina
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ABSTRACT |
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We evaluated the effects of
increasing the viscosity () in peritubular capillary perfusates
(PCP; 20 mM HNaPO4
-Ringer, pH 7.4) on proximal
convoluted tubule (PCT) acidification. Micropuncture experiments were
performed with simultaneous luminal and peritubular perfusion. Changes
in pH of a 20 mM HNaPO4
-Ringer (pH 7.4 at
t = 0) droplet placed in PCT lumen were measured with
H+-sensitive microelectrodes. By adding neutral
dextran (molecular wt 300,000-400,000) to the PCP,
was
increased. The effect of 10
5 M ATP added to normal-
PCP was evaluated. High
increased H+ flux (85 and 97%
when
was increased 20 and 30%, respectively, above the control
value). This increase was abolished by adding the nitric oxide
antagonist N
-nitro-L-arginine
(L-NNA; 10
4 M) or the purinoreceptor
antagonists suramin (10
4 M) and reactive blue 2 (3 × 10
5 M). Addition of 5 × 10
3 M
L-arginine to the peritubular perfusate overcame the
inhibitory effect of L-NNA on high-
-induced increase in
H+ flux. ATP increased H+ flux (80%), and this
effect was blocked by L-NNA. These results suggest that
changes in
can modulate proximal H+ flux, at least in
part, through ATP-dependent nitric oxide release from the endothelial
cells of the peritubular capillaries.
nitric oxide; shear stress; adenosine 5'-triphosphate antagonists; N-nitro-L-arginine; micropuncture; hydrogen ion secretion
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INTRODUCTION |
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OF THE RENAL BLOOD
FLOW, ~80% circulates through the renal cortex. The
peritubular capillaries stemming from the efferent arteriole form a
large net in close vicinity to the renal tubules. The formation of a
protein-free ultrafiltrate induces changes in the biophysical
properties of blood leaving the glomeruli (19). One such
property is viscosity (), which depends on the plasma protein
concentration and the hematocrit. Of the filtered load of volume and
sodium, 70-80% is reabsorbed from the proximal tubular lumen
toward the peritubular capillaries. The constancy of proximal fractional sodium and water reabsorption is called the
glomerular-tubular balance (GTB). This is a highly regulated process
and, under physiological conditions, is kept constant
(14). The interplay of hydrostatic and oncotic pressures
between peritubular capillaries and the interstitial space plays a
critical role in maintaining GTB (21).
Recently, the endothelium-dependent relaxing factor, which is thought
to be nitric oxide (NO) (22), was shown to be involved in
the regulation of HCO3 reabsorption (or
H+ secretion) at the proximal convoluted tubule (PCT) of
the rat kidney. Indeed, Wang (28), using micropuncture
techniques, showed an increase in HCO3
reabsorption
after NO formation. Simultaneously, we described a cGMP-dependent
mechanism that promotes H+ secretion in PCT by activating
Na+/H+ exchange. This mechanism is stimulated
by NO agonists added to peritubular capillary perfusates
(1). Furthermore, Green et al. (13) showed
that cGMP stimulates Na+/H+ exchange at the
renal brush border. On the other hand, most of the Na+
reabsorption in PCT depends on Na+/H+ exchange
(4).
In large arteries, carbamylcholine, bradykinin, and ATP stimulate NO
release and cGMP accumulation in endothelial cells (11). Another important agonist of NO release is shear stress (7, 23,
26). Moreover, shear stress also induces ATP release from endothelial cells (6, 7, 20). Between shear stress and the
rate of change in shear strain, is the constant of proportionality. The purpose of the present work was to address the question of whether
changes in fluid
(i.e., shear stress) in peritubular capillaries
could affect, by modifying NO release, proximal tubule H+
transport, through a paracrine mechanism.
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METHODS |
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Materials
H+ ionophore, cocktail A, was purchased from Fluka (Ronkonkoma, NY). Other chemicals were from Sigma (St. Louis, MO), ICN (Costa Mesa, CA), and Pharmacia (Uppsala, Sweden).General Procedure
Male Wistar rats, weighing ~300 g, were anesthetized with pentobarbital sodium (50 mg/kg ip) and prepared for micropuncture experiments as previously described (2). Briefly, the right jugular vein was cannulated, a cannula was placed in the trachea, and the left kidney was exposed through a flank incision. Peritubular capillaries were perfused with micropipettes made of 1.5-mm-outer-diameter borosilicate glass tubing (Hilgenberg, Malsfeld, Germany) that had a tip diameter of 5-10 µm. PCTs in the perfused area were impaled with double-barreled micropipettes, one barrel containing Sudan black castor oil and the other, the luminal perfusion solution. The oil column injected in the tubule lumen was split by a droplet of perfusion solution, and a single-barreled pH-sensitive microelectrode, positioned two to three loops downstream, was employed to continuously measure the pH of the droplet.pH Measurements
Liquid-membrane pH-sensitive microelectrodes were made as previously described (3) and had slopes of 54-58 mV/pH unit. Microelectrodes were calibrated in phosphate-Ringer buffer. The voltage difference between the pH microelectrode and a reference calomel electrode placed in contact with the skinned tip of the tail is proportional to the pH of the luminal fluid. Voltages were measured with a high-impedance electrometer (FD 223; World Precision Instruments, Sarasota, FL).Solutions
The control peritubular perfusate contained (in mM) 105 NaCl, 5 KCl, 1 CaCl2, 20 HNaPO4Theoretical Assumptions
During phosphate perfusion, H+ secretion results in acidification of the luminal solution and titration of alkaline phosphate. Therefore, acid phosphate concentration rises and reaches steady state. Detailed treatments of this model have been previously published (2, 8, 12). Using pH values recorded from microelectrode measurements, H2NaPO4 concentration at time t was calculated according to
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Experimental Groups
The luminal perfusion solution was the same for all the experimental groups, except for the Na+-free groups. The composition of peritubular capillary perfusion solutions varied as follows.Control. Peritubular capillaries were perfused with the peritubular perfusate solution described above as the control.
Increased -perfusion.
Four sets of experiments were performed, in which
in peritubular
capillary perfusates was increased 10, 20, and 30% over the normal
Ringer values of
by addition of neutral dextran (molecular wt
300,000- 400,000). In another experiment, Percoll (colloidal silica coated with polyvinyl pyrrolidone) was added to increase
30% over the control value. The alteration in osmolality of peritubular perfusion solution by the addition of dextran or Percoll was negligible. The final
relative to Ringer-phosphate solution was
measured with an Oswald viscosimeter at room temperature.
ATP.
ATP (105 M) was added to the peritubular perfusion solution.
Antagonists of purinergic receptors.
Suramin (104 M) or 3 × 10
5 M reactive
blue 2 (RB2) was added to the high-
(
increased 30% by dextran)
peritubular perfusate. Suramin is a nonselective
P2-purinoreceptor antagonist (9), and RB2 is a
more selective P2 type-Y-purinoreceptor antagonist (10).
Antagonist of NO.
N-nitro-L-arginine
(L-NNA; 10
4 M) was added to the high-
(
increased 30% by dextran) or the ATP-containing peritubular perfusate. L-NNA inhibits the synthesis of NO, acting as a competitive
antagonist of L-arginine, the substrate of NO synthase
(15, 17, 27).
L-Arginine+L-NNA+high .
L-Arginine (5 × 10
3 M) was added to the
high-
(
increased 30% by dextran) perfusate containing
L-NNA.
Na+ free.
Composition of Na+-free peritubular and luminal solutions
is detailed above. Effect of high in Na+-free
experiments was evaluated by increasing
30% in peritubular Na+-free perfusate by adding dextran.
Statistics
Results are shown as means ± SE. Data were evaluated with one-way analysis of variance, and multiple comparisons including all experimental groups were performed with a Student-Newman-Keuls test. ![]() |
RESULTS |
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Kinetics of Proximal Luminal Acidification in Different Experimental Groups
The steady-state pH was the same in all groups studied. The acidification rate constant increased by increasing
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Effects of Changes in of the Peritubular Perfusate on PCT
H+ Flux
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Effects of ATP on PCT H+ Flux
Addition of 10
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Effects of Purinergic-Receptor Antagonists on Increased PCT H+ Flux Induced by Dextran
The presence of antagonists of P2 purinoreceptors in peritubular perfusates prevented the high-
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Effect of Na+-Free Solutions on H+ Flux in PCT
Net H+ flux was significantly reduced with Na+-free peritubular and luminal perfusion. In such conditions, H+ flux was 0.125 ± 0.010 nmol · cm ![]() |
DISCUSSION |
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NO synthesis is involved in the control of kidney hemodynamic and tubular functions (16). Endothelial cells affect the function of epithelial cells by a paracrine-like mechanism involving the release of NO (1, 18, 27, 28). Moreover, NO induces an increase in proximal tubule acidification, which depends on cGMP production in proximal tubule epithelial cells (1, 28).
A consequence of glomerular filtration is an increase in the
concentration of plasmatic proteins and hematocrit in the efferent arteriole. With a filtration fraction of 30%, the hematocrit will increase from 40 to 50% and the apparent of blood would increase ~30% (29). We increased the
of the peritubular
perfusate solution in this range. In the present work, we found that an
increase in the
of the peritubular capillary perfusate induces an
increase in proximal tubule acidification. This effect appears to be
independent of the agent used to increase flow
, because peritubular
perfusates containing either dextran or Percoll evoked the same
response. The effect of high
was observed when the increase in
was >20%. An increase in
of 10% was without effect. The
-induced increase in PCT H+ flux was blocked by
L-NNA, suggesting that it depends on NO release. L-NNA not only abolished the effect of dextran but also
inhibited H+ flux below the control value. This result is
coincident with previous observations, where we found that
10
4 M L-NAME decreased H+ flux
37% below the control value (1). These results suggest that there is a basal NO-dependent H+ flux. Shear stress is
directly related to
and is a strong agonist of NO release in large
arteries (7, 11, 23, 26). Moreover, the inhibitory effect
of L-NNA on H+ flux stimulated by high
was
abolished by L-arginine. This result gives support to the
hypothesis that NO release was involved in the modulation of
H+ flux by
. Furthermore, the effect of high
was
absent in Na+-free conditions, suggesting that the
Na+/H+ exchanger is involved in the
high-
-induced increase in H+ flux. On the other hand,
the effect of increased
was abolished by
P2-purinoreceptor blockade with suramin and RB2. Several
subtypes of P2 purinoreceptors have been characterized.
Generally, vascular P2X purinoreceptors are located on
smooth muscle to mediate contraction, whereas P2Y
purinoreceptors, present on endothelial cells and on smooth muscle, are
involved in vasodilatation (24). Although not highly
specific, RB2 seems to be more selective for P2Y
(10); thus the effect of high
could be mediated, at
least in part, by these receptors. ATP is a strong agonist of NO
release from endothelial cells of large arteries (11). It
has been shown that ATP is released from the endothelium by shear
stress (6, 20). Therefore, changes in
could modify NO
synthesis through ATP release. However, we cannot exclude that shear
stress per se could stimulate NO release directly through activation of
another kind of mechanism (5). In the present work, the
peritubular perfusate containing 10
5 M ATP induced an
increase in H+ flux similar to that observed with dextran
or Percoll. The effect of ATP resembled the effect previously found
with bradykinin and carbamylcholine (1) and was also
blocked by L-NNA, suggesting an involvement of NO release.
Our results agree with those reported by Wang (28), who
found an increase in HCO3 flux in PCT induced by
10
6 M sodium nitroprusside (SNP), a NO donor. However,
Linas and Repine (18) reported experiments, performed on
cocultures of endothelial and epithelial cells, in which cGMP
production induced by endothelium-derived NO decreased apical
Na+/H+ exchange. Also, in rabbit proximal
tubule cells, NO stimulated soluble guanylate cyclase and caused
inhibition of Na+/H+ exchange
(25). Although we do not have a clear explanation for this
discrepancy, there are at least two possible causes. First, we, like
Wang (28), used an in vivo model, which implies a specific
geometry between both structures, the epithelial cells of the proximal
tubule and the underlying endothelium. Second, and perhaps more
importantly, in our model the exposure to the agonists lasts a short
time, no longer than 3 min, which, however, was long enough to perform
H+ flux measurements. Na+ flux assessment, as
measured by Linas and Repine (18), required longer
exposure to the agonists. We did not subject our preparation to more
prolonged NO exposure, which may have effects different from those
reported here. On the other hand, Roczniak and Burns (25) used 1 mM SNP concentration. Wang (28)
found that SNP has dual effects depending on its concentration,
stimulating HCO3
reabsorption at low (1 µM) and
inhibiting it at high concentration (1 mM).
In summary, NO produced by increased stimulates the synthesis of
cGMP in tubular epithelial cells, activating the
Na+/H+ exchanger, and thus coupling filtered
Na+ load to proximal tubule Na+ and water
reabsorption. The present results suggest that a mechanism of this type
could contribute, in addition to the Starling forces in the peritubular
capillaries, to the control of GTB.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Altamirano for a careful reading of the manuscript and valuable suggestions and A. Müller for superb technical assistance.
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FOOTNOTES |
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This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas Grants 2851-95851 and 2851-133521.
Address for reprint requests and other correspondence: C. Amorena, M. T. de Alvear 2270, 1122 Buenos Aires, Argentina (E-mail: Carlos.Amorena{at}unsam.edu.ar).
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. Section 1734 solely to indicate this fact.
Received 17 December 1999; accepted in final form 19 October 2000.
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