Peritubular AVP regulates bicarbonate reabsorption in cortical distal tubule via V1 and V2 receptors

Raif Musa-Aziz1, Maria Luisa Morais Barreto-Chaves2, and Margarida De Mello-Aires1

Departments of 1 Physiology and Biophysics and 2 Anatomy, Instituto de Ciências Biomédicas, University of São Paulo, São Paulo 05508-900, Brazil


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

10.1152/ajprenal.00056.2001. Peritubular arginine vasopressin (AVP) regulates bicarbonate reabsorption in the cortical distal tubule via V1 and V2 receptors. The dose-dependent effects of peritubular AVP on net bicarbonate reabsorption (JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) were evaluated by stationary microperfusion of in vivo early (ED; distal convoluted tubule) and late distal (LD; connecting tubule and initial collecting duct) segments of rat kidney, using double-barreled H+-sensitive, ion-exchange resin/reference (1 M KCl) microelectrodes. AVP (10-11 M) perfused into peritubular capillaries increased JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, compared with basal levels during intact capillary perfusion with blood, in ED and LD segments. AVP (10-9 M) also increased JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in both segments, but the effect of AVP (10-11 M) was significantly higher. A specificV1-receptor antagonist alone or with AVP (10-11 or 10-9 M) reduced JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> below basal levels. A specific V2-receptor antagonist alone or plus AVP (10-11 M) did not affect JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> but increased AVP (10-9 M)-mediated stimulation. 8-Bromoadenosine 3',5'-cyclic monophosphate alone reduced JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> below basal levels and also reduced AVP (10-11 M)-mediated stimulation. (Deamino-Cys1, D-Arg8) vasopressin (a V2-selective agonist) also reduced JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> below basal levels. These results show that peritubular AVP stimulates JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in ED and LD segments via basolateral V1 receptors and that basolateral V2 receptors have a dose-dependent inhibitory effect mediated by cAMP. The data also indicate that endogenous AVP stimulates distal JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> via basolateral V1 receptors.

arginine vasorpressin; distal bicarbonate reabsorption


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CORTICAL DISTAL TUBULE of mammalian kidney plays an important role in the renal control of H+ secretion and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. Processes such as electrogenic H+ secretion and electroneutral Na+/H+ exchange, as well as H+-K+-ATPase and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and Cl- channels, play a key role in determining the rate and direction of bicarbonate transport in this nephron segment (9).

Data from our laboratory showed that luminal arginine vasopressin (AVP; 10-9 M) stimulates the Na+/H+ exchanger in early distal (ED; distal convoluted tubule) and late distal (LD; connecting tubule and initial collecting duct) segments, as well as the vacuolar H+-ATPase in LD segments, via activation of V1 receptors (3), confirming previous studies showing that AVP stimulates proton secretion in the distal tubule (5, 17) and cortical collecting duct (5, 26). However, the nature of the mechanism underlying AVP action on distal nephron bicarbonate reabsorption is not yet clearly defined because, in A6 cells (an amphibian distal nephron cell line), the addition of AVP either at low (10-10 M) or at high (10-6 M) concentrations inhibits the basolateral Na+/H+ exchanger activity (10). Besides this, studies in thick ascending limbs showed that AVP stimulates the basolateral, while inhibiting the apical, Na+/H+ antiporter (25).

In addition, most studies have detected AVP action when it is applied at the distal nephron basolateral surface, mediated mostly by V2 receptors via the adenylate cyclase/cAMP-protein kinase A signaling system. This pathway, at high-AVP concentrations, is expected to inhibit the Na+/H+ exchanger (6). However, in recent years V1 receptors have been detected in both apical and basolateral membrane domains and have been shown to mediate AVP activity via phospholipase C-inositol 3,4,5-triphosphate (IP3)-calcium signaling (14, 15, 23). On the other hand, it is known that protein kinase C, via phosphorylation, may stimulate the Na+/H+ exchanger (11). Recently, we have shown that the stimulatory effect of AVP on the net rate of Na+-dependent intracellular pH (pHi) recovery in Madin-Darby canine kidney (MDCK) cells, a cell line with many morphological and physiological similarities to the mammalian distal nephron, is via activation of V1 receptors located on the basolateral cell membrane surface and that the basolateral V2 receptors have a dose-dependent inhibitory effect (22). Thus, with the consideration that the response of distal bicarbonate reabsorption to AVP may vary with the hormonal doses being studied and with the type of receptor present on the distal cell membrane surface, the present study was designed to determine whether peritubular AVP at either low (10-11 M) or at high (10-9 M) concentrations regulates bicarbonate transport in the rat cortical distal tubule. For this purpose, we evaluated the kinetics of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption by the stationary microperfusion technique in in vivo ED and LD segments. To detect which specific basolateral receptor AVP regulates distal bicarbonate reabsorption, we also examined the effects of the V1 or V2 receptor-specific antagonists 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; a membrane-permeant cAMP analog) and (deamino-Cys1, D-Arg8) vasopressin (dDAVP; a V2-selective agonist) on this process.

Our results indicate that peritubular AVP stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in ED and LD segments via basolateral V1 receptors and that basolateral V2 receptors have a dose-dependent inhibitory effect mediated by cAMP. This dual regulation of bicarbonate reabsorption may represent a relevant mechanism in conditions of volume depletion or plasma osmolality increase. In addition, our data indicate that endogenous AVP stimulates bicarbonate reabsorption in ED and LD segments via basolateral V1 receptors.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Male Wistar rats, weighing 200-340 g and obtained from the Instituto de Ciências Biomédicas, were anesthetized with Inactin (100 mg/kg ip; Byk-Gulden, Konstanz, Germany). They received a rat pellet diet and water ad libitum until the time of the experiment. The rats were prepared for in vivo micropuncture as described previously (18). The left jugular vein and left carotid artery were cannulated for infusions and blood withdrawal, respectively. A tracheostomy was performed. The kidney was isolated by a lumbar approach and immobilized in situ by Ringer-agar in a Lucite cup. During the experiment, the rats received venous saline containing 3% mannitol at 0.05 ml/min.

Bicarbonate reabsorption in ED and LD segments was calculated from the continuous measurement of luminal pH by means of microelectrodes in a fluid column isolated by castor oil in the tubule lumen and after the intratubular pH changes toward the steady-state level (12, 18). The microperfusion procedure involved impalement of a proximal loop with a double-barreled micropipette made from theta glass tubing (R&D Optical Systems, Spencerville, MD). One barrel was filled with Sudan black castor oil, and the other was filled with the luminal perfusion solution colored with 0.05% FD&C green. Initially, the luminal perfusion was used to detect ED or LD loops. A double-barreled microeletrode was then inserted into the ED or LD loop. Afterward, luminal perfusion was performed at a rate sufficient to elevate luminal distal tubular loop pH to near that of the original perfusion solution (pH = 8). Then, a column of oil was injected into the proximal tubular lumen, blocking the flow of fluid. Intratubular pH was measured as the voltage difference between the two barrels of the microeletrode made from Hilgenberg (Malsfeld, Germany) double-barreled asymmetric glass capillaries. The larger barrel contained a H+-sensitive ion-exchange resin (Fluka, Buchs, Switzerland), and the smaller one contained 1 M KCl colored by FD&C green (reference barrel). Transepithelial electrical potential difference was the difference between the reference barrel and ground. This parameter was an additional criterion for the recognition of ED or LD segments (13). Intratubular pH changes were recorded continuously in the same tubule with a Beckman model RP dynograph and digitized by a Dell 333D microcomputer equipped with an analog-to-digital conversion board (Lynx, São Paulo, Brazil) for data acquisition and processing.

The luminal pH fell from its initial value of eight toward the stationary level. Along this curve, intratubular concentrations of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were calculated from intratubular pH values and from the systemic PCO2 by use of the Henderson-Hasselbalch equation at intervals of 1 s, because we have previously demonstrated that renal cortical PCO2 is similar to that of arterial blood (21). The rate of tubular acidification was expressed as the half-time of the reduction in the injected HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> levels to their stationary level (t1/2). Net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption (JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) was calculated from the equation
J<SUB>HCO<SUP><IT>−</IT></SUP><SUB>3</SUB></SUB><IT>=k</IT>([HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>i</SUB><IT>−</IT>[HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>s</SUB>)<IT>r/</IT>2
where k is the rate constant of the reduction in luminal bicarbonate [k = ln2/(t1/2)], r is the tubule radius, and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]s are the concentrations of the injected HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at the stationary level, respectively.

The pH microeletrodes were calibrated before and after every impalement on the kidney's surface by superfusion with 20 mM phosphate-Ringer buffer solutions containing 130 mM NaCl at 37°C. The pH values were adjusted to 6.5, 7.0, and 7.5 with 0.1 N NaOH or HCl.

The luminal control perfusion solution contained (in mM) 100 NaCl, 25 NaHCO3, 5 KCl, 1 CaCl2, and 1.2 MgSO4. The osmolality was adjusted to 300 mosmol/kgH2O with raffinose.

In each tubule, several measurements of JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were first done during intact capillary perfusion with blood (basal values), next in the presence of the capillary perfusion solution (experimental values), and then again during capillary perfusion with blood (recovery of basal levels).

Peritubular capillaries were perfused with single pipettes made of thin-walled glass tubes (Kimax; 1.5- to 2.5-mm OD), having a tip diameter of 10 µm. Air pressure (1-1.2 atm) was applied to these pipettes for peritubular capillary microperfusion. This perfusion was considered satisfactory when the perfused area reached two or three loops beyond the luminally perfused distal segment. Peritubular capillary perfusion was performed with a solution containing (in mM): 140 NaCl, 20 NaHCO3, 5 KCl, 1 CaCl2, 1.2 MgSO4, and 5 Na+-acetate at pH 7.4. This solution was preequilibrated with 5% CO2 in air. The experimental groups of peritubular capillary perfusion solution studied were selected to combine several possibilities of peritubular drug administration, i.e., AVP (10-11 or 10-9 M) alone or plus anti-V1 or anti-V2 (10-5 M), 8-Br-cAMP (10-4 M) alone or plus AVP (10-11 M), and dDAVP (10-9 M). In each tubule, only one peritubular perfusion solution was used.

AVP (mol wt 1.084), V1-receptor-specific antagonist [anti-V1; (beta -mercapto-beta , beta -cyclopentamethylene-propionyl1, O-Me-Tyr2, Arg8) vasopressin; (MCMV)] (20), V2-receptor-specific antagonist [anti-V2, (adamantaneacetyl1, O-Et-D-Tyr2, Val4, aminobutyryl6, Arg8,9) vasopressin] (16, 20), 8-Br-cAMP, dDAVP [(deamino-Cys1, D-Arg8) vasopressin], as well as all other applied chemicals were obtained from Sigma, St. Louis, MO.

The pH and PCO2 in samples of blood collected from the carotid artery were measured with a Radiometer ABL 5 blood-gas system. During the experiments, urine flow and Na+ excretion were also measured. Na+ in urine collected from urinary bladder was measured by flame photometry.

The data are shown as means ± SE. Statistical comparisons among parameters of intact, experimental, and postexperimental capillary perfusions performed within the same experimental group were made by using a t-test. Differences among experimental groups were evaluated by analysis of variance (1-way) with contrasts by using the Bonferroni technique, where n is the number of animals (mean of several measurements) when urine or blood collection was performed or the number of perfused tubules (mean of several perfusions).


    RESULTS
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ABSTRACT
INTRODUCTION
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DISCUSSION
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To detect whether the doses of AVP and V1- or V2-receptor antagonists used during capillary microperfusion would cause an effect on renal function and to follow acid-base conditions of the rats, we measured urine flow, Na+ excretion, and systemic acid-base parameters. During capillary microperfusion with these agents, these data were similar to the basal values found during capillary perfusion with blood [urine flow = 0.147 ± 0.018 ml · min-1 · kg-1 (18 measurements, 6 animals); urinary sodium excretion = 8.07 ± 0.85 µeq · min-1 · kg-1 (18 measurements, 6 animals); pH = 7.35 ± 0.12; PCO2 = 35.3 ± 0.84 Torr and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> = 24.2 ± 0.73 mM (59 measurements, 59 animals)].

In both ED and LD segments, no statistical differences were observed between the transepithelial electrical potential differences obtained in all peritubular capillary perfusion groups and the basal values found during capillary perfusion with blood [ED segments -16.9 ± 1.58 mV (222 measurements, 93 tubules) and LD segments -48.3 ± 4.49 mV (227 measurements, 100 tubules)].

In both ED or LD segments, intratubular steady-state pH values found during capillary microperfusions in all experimental groups were not significantly different from basal data obtained during capillary perfusion with blood [ED segments = 6.99 ± 0.55 (n = 222 measurements and 93 tubules) and LD segments = 7.05 ± 0.63 (n = 227 measurements and 100 tubules)], indicating that during peritubular capillary perfusion with an artificial solution at physiological pH and PCO2, tubular acidifying capacity was maintained at normal levels.

The mean half-time of the reduction in luminally injected HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> levels to their stationary level (t1/2) is given in Table 1 (ED segments) and Table 2 (LD segments).

                              
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Table 1.   Half-time of reduction in luminally injected HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP> in early distal tubule during capillary perfusion with a control solution or different agents


                              
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Table 2.   Half-time of reduction in luminally injected HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP> in late distal tubule during capillary perfusion with a control solution or different agents

Figure 1 shows the sequence of perfusion in four different ED segments to which the control solution or AVP (10-11 M) was applied during peritubular capillary perfusion. During capillary perfusion with the control solution, it is apparent that over a period of several minutes no significant change in bicarbonate reabsorption occurs compared with basal levels during intact capillary perfusion with blood (Fig. 1A), confirming the capacity of the distal tubule to maintain an adequate rate of acidification during capillary perfusion with an artificial solution at physiological pH and PCO2. It is clear that AVP (10-11 M) added to the capillary solution significantly stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsoption (Fig. 1B). The addition of anti-V1 to capillary perfusion reduces AVP (10-11 M)-mediated stimulation below basal blood perfusion levels (Fig. 1C). These data indicate that endogenous AVP stimulates distal bicarbonate reabsorption via V1 receptors and that the capillary administration of AVP (10-11 M) increases this process. However, the addition of anti-V2 does not affect AVP (10-11 M)-mediated stimulation (Fig. 1D). It is also clear that, in all of these situations, recovery of basal levels occurs after the experimental capillary perfusion. Figures 2 and 3 give mean data of bicarbonate reabsorption in ED and LD segments to which these experimental solutions were applied in the capillary perfusion.


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Fig. 1.   Sequence of measurements of net bicarbonate reabsorption (JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) in 4 different early distal (ED) tubular segments. In each tubule, several measurements of JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were done first during initial peritubular capillary perfusion with blood (basal values), then in the presence of experimental capillary perfusion, and then again during capillary perfusion with blood (recovery of basal levels). Experimental capillary perfusions were performed with a control solution (A), arginine vasopressin (AVP; 10-11 M) alone (B), AVP (10-11 M) plus anti-V1 (10-5 M; C), and AVP (10-11 M) plus anti-V2 (10-5 M; D).



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Fig. 2.   Effect of control solution, anti-V1, or anti-V2 (10-5 M) peritubular capillary perfusion on JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in ED (A) and late distal (LD; B) tubular segments. Values are means ± SE; n = no. measurements/no. of tubules (in parentheses). No statistical differences were observed between the perfusion with control solution and the respective peritubular capillary perfusions with blood. No statistical differences were observed between initial peritubular capillary perfusion with blood (basal values) and the respective postexperimental blood capillary perfusion (recovery of basal levels). *P < 0.01 compared with the respective intact peritubular capillary perfusion with blood.



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Fig. 3.   Effect of AVP (10-11 M) peritubular capillary perfusion on JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in ED (A) and LD (B) tubular segments. Capillary perfusions were performed with AVP (10-11 M) alone or plus anti-V1 or anti-V2 (10-5 M). Values are means ± SE; n = no. of measurements/no. of tubules (in parentheses). No statistical differences were observed between capillary perfusion with blood (basal values) and the respective postexperimental capillary perfusion groups (recovery of basal levels). *P < 0.01 compared with the respective capillary perfusion with blood.

Figure 4 shows that the capillary administration of AVP (10-9 M) also increases ED and LD HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. The addition of Anti-V1 to peritubular perfusion reduces AVP (10-9 M)-mediated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption below basal blood perfusion levels in ED and LD segments, confirming that endogenous AVP stimulates distal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption via V1 receptors. On the other hand, when AVP (10-9 M) is given together with anti-V2, the effect on bicarbonate reabsorption is significantly greater than with AVP (10-9 M) alone in ED and LD segments, indicating that V2 receptors have a dose-dependent inhibitory effect. Also, in these situations, recovery of basal levels is observed after the experimental capillary perfusions.


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Fig. 4.   Effect of AVP (10-9 M) capillary perfusion on JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in ED (A) and LD (B) tubular segments. Capillary perfusions were performed with AVP (10-9 M) alone or plus anti-V1 or anti-V2 (10-5 M). Values are means ± SE;. n = no. of measurements/no. of tubules (in parentheses). No statistical differences were observed between capillary perfusion with blood (basal values) and the respective postexperimental capillary perfusion groups (recovery of basal levels).*P < 0.01 compared with the respective capillary blood-perfusion group. $P < 0.01 compared with AVP (10-9 M)-alone group.

Figure 5 gives the effect of the addition of 8-BrcAMP (10-4 M; a membrane-permeant cAMP analog) to the experimental peritubular capillary perfusion in ED and LD segments. 8-BrcAMP alone reduces JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> below basal levels and also reduces AVP (10-11 M)-mediated stimulation, showing an inhibitory effect mediated by cAMP.


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Fig. 5.   Effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 10-4 M) peritubular capillary perfusion on JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in ED (A) and LD (B) tubular segments. Capillary perfusions were performed with AVP (10-11 M) alone or 8-Br-cAMP (10-4 M) alone or plus AVP (10-11 M). Values are means ± SE; n = no. of measurements/no. of tubules (in parentheses). No statistical differences were observed between capillary perfusion with blood (basal values) and the respective postexperimental capillary perfusion groups (recovery of basal levels). *P < 0.01 compared with the respective capillary blood-perfusion group. @P < 0.05 compared with the respective capillary blood-perfusion group. &P < 0.01 compared with AVP (10-11 M)-alone group.

Figure 6 summarizes the previous results obtained in ED and LD segments. The effect of 10-11 M AVP was higher than that of 10-9 M AVP. However, when anti-V2 is given together with 10-9 M AVP, bicarbonate reabsorption increases toward the 10-11 M AVP values. On the other hand, in the presence of 8-BrcAMP the effect of 10-11 M AVP is similar to the effect of 10-9 M AVP alone. These results indicate that the stimulation of V2 receptors has a dose-dependent inhibitory effect mediated by cAMP.


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Fig. 6.   Comparison between the effects of 10-11 and 10-9 M AVP capillary perfusions on JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in ED (A) and LD (B) tubular segments. Values are means ± SE; n = no. of measurements/no. of tubules (in parentheses). &P < 0.01 compared with AVP (10-11 M)-alone group. #P < 0.01 compared with AVP (10-11 M) plus anti-V2 (10-5 M) group. $P < 0.01 compared with AVP (10-9 M)-alone group.

Confirming this hypothesis, Fig. 7 shows that dDAVP (10-9 M; an AVP analog that specifically binds to adenylyl cyclase-coupled V2 receptors) reduces HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption below basal blood perfusion levels, in both ED and LD segments.


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Fig. 7.   Effect of (deamino-Cys1, D-Arg8) vasopressin (dDAVP; 10-9 M) peritubular capillary perfusion on JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in tubule. A: sequence of measurements in 1 ED tubular segment, first during initial peritubular capillary perfusion with blood (basal values), then in the presence of dDAVP capillary perfusion, and then again during capillary perfusion with blood (recovery of basal levels). B: mean data of JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in ED (a) and LD (b) segments to which dDAVP was applied in the capillary perfusion. Values are means ± SE; n = no. of measurements/no. of tubules (in parentheses). No statistical differences were observed between capillary perfusion with blood (basal values) and the respective postexperimental capillary perfusion groups (recovery of basal levels). *P < 0.01 compared with the respective capillary blood-perfusion group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The cortical distal tubule of the mammalian kidney has an important role in the renal control of H+ secretion and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption (9). However, the nature of the mechanism underlying AVP action on distal nephron bicarbonate reabsorption is not yet clearly defined. Considering the possible existence of a dose-dependent hormonal effect (22) and of functional basolateral V1 and V2 AVP receptors (29), in the present work we studied the effect of peritubular AVP (10-11 or 10-9 M) on the kinetics of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in both ED and LD segments. We examined the effect of peritubular capillary perfusion of this hormone in vivo by using a stopped-flow microperfusion technique, which is not affected by glomerular filtration rate. Our results indicated that this procedure also avoids systemic alterations caused by hormone infusion, confirmed by an absence of changes in urine flow, Na+ excretion, and systemic acid-base values during experiments where peritubular capillary perfusion was performed. Furthermore, the luminal perfusion solution contains raffinose (a nonreabsorbable molecule) to reach isotonicity so as to prevent fluid reabsorption induced by the hormone. Another advantage of peritubular capillary perfusion is to ascertain that the measurements of acidification kinetics are performed at well-determined hormonal levels, because it is known that some peptides have a short half-life when injected parenterally. This is important because an intact structure of AVP is required for binding to the receptor (2).

In all groups studied, in ED and LD segments during the recovery period after experimental capillary perfusion, a recovery of basal levels of the bicarbonate reabsorption was observed, indicating that the substitution of peritubular capillary blood by artificial solutions appears to be a valid method for the study of factors affecting tubular acidification. Furthermore, repeated luminal perfusion procedures did not significantly alter the transepithelial electrical potential difference, intratubular steady-state pH values, and JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 1), indicating that sequential comparisons in the same nephron segment can be used to accurately assess the effect of experimental capillary perfusions. This represents a considerable experimental advantage, because it permits paired measurements of basal and experimental conditions in the same nephron segment.

In addition, no significant differences were observed between intratubular steady-state pH values of capillary-perfused groups and the respective control groups. Changes in this parameter are related to several factors, including the function of H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters. These findings suggest that the main driving force for H+ secretion across the apical membrane was not altered during the experiments with peritubular capillary perfusion, indicating that during peritubular capillary perfusions at physiological pH and PCO2, tubular pH-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> gradients were maintained at normal levels and modification of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption rates were only due to changes in the rate (t1/2) at which these gradients were reached.

Our results indicate that peritubular AVP (10-11 or 10-9 M) has a direct effect, stimulating HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in ED and LD segments, as expressed by a significant fall in acidification half-time and consequent increase in JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Tables 1 and 2, Figs. 3 and 4). The present demonstration of AVP-induced enhancement of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption by distal tubules is not the first study of this kind, because in vitro studies in rat cortical collecting duct (26) and rat distal tubules (17) showed AVP-stimulated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption.

Our results show that the addition of anti-V1 to peritubular perfusion reduces AVP (10-11 or 10-9 M)-mediated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in ED and LD segments, indicating that peritubular AVP stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in both segments via basolateral V1 receptors. These data are in accordance with the identification of V1 receptors for AVP in the distal nephron, especially in the cortical portion of the collecting duct (15, 29). These data are also compatible with studies showing that V1 receptors mediate AVP action mostly via a Gq11 protein-phospholipase C-IP3-protein kinase C-Ca2+ pathway (7, 8, 15, 28) and that protein kinase C may stimulate the Na+/H+ exchanger (3, 11, 22).

At high concentrations, AVP is known to interact with V1 receptors, causing the liberation of arachidonic acid, which is part of a path that elevates cell calcium and, consequently, may inhibit the Na+/H+ exchanger (24). This inhibitory effect of a high concentration of AVP via V1 stimulation is apparent in our present data in ED and LD segments, which show that the stimulatory effect of peritubular 10-11 M AVP plus anti-V2 on distal bicarbonate reabsorption is higher than with 10-9 M AVP plus anti-V2 (Fig. 6).

It is well known that V2 receptors mediate a dose-dependent adenylate cyclase-cAMP-protein kinase A pathway that, at high-AVP concentrations, is expected to inhibit the Na+/H+ exchanger (6). This behavior is compatible with our present data in ED and LD segments, showing that a V2-receptor antagonist plus 10-11 M AVP does not affect JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> but the addition of 10-9 M AVP increases JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to near the levels observed when 10-11 M AVP alone is given (Fig. 6). These data indicate that, in presence of 10-11 M AVP, the cAMP levels are too low to inhibit the Na+/H+ exchanger. These results also show that the inhibitory effect of a high concentration of AVP was prevented by simultaneous capillary perfusion with the anti-V2 agent. In addition, our present results show that with 8-BrcAMP (a cAMP analog) the effect of 10-11 M AVP is similar to the effect of 10-9 M AVP alone (Fig. 6), confirming that in presence of 10-11 M AVP alone the cAMP levels are too low to inhibit the Na+/H+ exchanger. These data also indicate that V2 receptors have a dose-dependent inhibitory effect mediated by cAMP. This hypothesis is confirmed by our results showing that dDAVP (an AVP analog that specifically binds to adenylyl cyclase-coupled V2 receptors) reduces HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption below basal levels in both ED and LD segments. These data agree with previous results from our laboratory showing that the stimulatory effect of AVP on the net rate of Na+-dependent pHi recovery in MDCK cells (a cell line with many morphological and physiological similarities to the mammalian distal nephron) is via activation of basolateral V1 receptors and that the basolateral V2 receptors have a dose-dependent inhibitory effect (22). However, the inhibitory effects of V2 receptors described here are at variance with previous data by Bichara et al. (5), who showed that systemic infusion of the V2 agonist dDAVP stimulates bicarbonate reabsorption in rat cortical distal tubules. This discrepancy could be explained because in their study (5) bicarbonate transport was measured during free-flow conditions in the distal tubule. In this situation, dDAVP infusion enhanced water reabsorption along the distal tubule (5), leading to luminal concentration of AVP that could, in turn, secondarily stimulate bicarbonate reabsorption by tubular cells. In this regard, we have previously demonstrated that 10-9 M luminal AVP stimulates the Na+/H+ exchanger and H+-ATPase in the cortical distal tubule via luminal V1 receptors (3). Our present results also show that the presence of the V2-receptor antagonist alone in peritubular perfusion has no effect on bicarbonate reabsorption in ED and LD segments (Fig. 2). These results indicate that in basal conditions the basolateral V2 receptors are not stimulated, confirming that these receptors have a hormonal dose-dependent inhibitory effect. This dual regulation of bicarbonate reabsorption may represent a relevant regulatory mechanism that prevents blood alkalinization in conditions of volume depletion, because it is known that plasma AVP usually reaches levels 20-30 times greater than normal when blood volume is reduced by 20-30% (in rats, dogs, and humans) or plasma osmolality is increased by 10% (4).

The effect of AVP during peritubular capillary perfusion raises the question of the distribution of receptors for this peptide on the cell surface. It appears reasonable to believe that the effect of AVP during capillary perfusion occurs due to the presence of V1 and V2 receptors at the distal cell basolateral membrane surface, because it is improbable that in tight epithelia such as the distal tubule this peptide could reach the luminal membrane via the paracellular shunt path during peritubular perfusion. In addition, our present data confirm earlier findings showing that, in rabbit cortical collecting duct, electrical responses to AVP in the bath are a composite of basolateral V1 and V2 receptor-mediated actions (29).

The possible physiological role of peritubular AVP action should also be considered. Our results show that the addition of V1-receptor antagonist alone or with 10-11 or 10-9 M AVP to peritubular capillary perfusion reduces bicarbonate reabsorption in ED and LD segments below the levels found during blood capillary perfusion (Figs. 1-4). This could be due to an unspecific inhibitory action of this V1-receptor antagonist, but it might also indicate that, in basal conditions, a basal level of AVP binding to peritubular V1 receptors may exist, causing some tonic activation of bicarbonate reabsorption. Inhibitory action of this nature has been reported before by Amorim and Malnic (1), demonstrating that the V1-antagonist MCMV (the same used by us in the present experiments) reduces distal K+ secretion in Wistar rats when given alone. To eliminate the possibility of an unspecific inhibitory effect of the V1-antagonist MCMV, these authors performed experiments in homozygous Brattleboro rats [which are known to be devoid of endogenous AVP production (27)], showing that this anti-V1 agent had no AVP-independent action, despite the presence of AVP receptors in these rats. On the basis of these findings, we may conclude that in basal conditions endogenous AVP stimulates bicarbonate reabsorption in ED and LD segments. On the other hand, our data show that there was no difference between the bicarbonate reabsorption of the basal group (in presence of intact capillary perfusion with blood) and the control solution capillary perfusion group (Fig. 2), which is apparently incompatible with the concept of endogenous AVP-stimulated bicarbonate reabsorption. However, an additional question involves the duration of AVP binding to its receptors in distal tubule basolateral membranes. Figure 1B indicates that AVP washout (during the recovery period in presence of capillary perfusion with blood) is not immediate but that it takes several minutes for levels to approach preexperimental capillary perfusion basal levels (during intact capillary perfusion with blood), suggesting that the maintenance of residual levels of AVP binding in basal conditions might be expected in the presence of capillary perfusion with a control solution. In addition, Figs. 1A and 2 demonstrate that, during capillary perfusion with the control solution, bicarbonate reabsorption does decrease somewhat but not significantly. Taken together, these findings support the view that, in basal conditions, a portion of normally observed distal bicarbonate reabsorption may be dependent on endogenous AVP levels.

In conclusion, we have undertaken in vivo stationary microperfusion studies in ED and LD segments of rat cortical tubule to evaluate the effects of peritubular capillary AVP (10-11 and 10-9 M) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. Our results indicate for the first time that peritubular AVP significantly stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in ED and LD segments via activation of basolateral V1 receptors and that basolateral V2 receptors have a dose-dependent inhibitory effect mediated by cAMP. In addition, our data indicate that endogenous AVP stimulates bicarbonate reabsorption in ED and LD segments of rat kidney via basolateral V1 receptors.


    ACKNOWLEDGEMENTS

We thank Dr. Gerhard Malnic, University of São Paulo, Brazil, for a careful reading of the manuscript and for helpful suggestions.


    FOOTNOTES

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-PADCT and Pronex). Portions of this work were presented at the Annual Meeting of the American Society of Nephrology, Toronto, Ontario, Canada, October 2000, and were published in abstract form (J Am Soc Nephrol 11: A0034, 2000).

Address for reprint requests and other correspondence: M. de Mello-Aires, Dept. of Physiology and Biophysics, Instituto de Ciências Biomédicas, Univ. of São Paulo, Av. Professor Lineu Prestes, 1524, SP 05508-900, Brazil (E-mail: mmaires{at}fisio.icb.usp.br).

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.

10.1152/ajprenal.00056.2001

Received 20 February 2001; accepted in final form 29 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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