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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

                              
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Table 1.   Perfusate composition in mM

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 2.   Bicarbonate reabsorption by distal tubules of animals perfused with solution 1 (5 mM HCO3, 40 mM Cl-)

                              
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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-)

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)

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, dagger  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.

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 5.   Bicarbonate reabsorption by distal tubules of animals perfused with solution 4 (5 mM HCO3 without Cl-)

                              
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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, dagger  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.

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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Capasso, G., R. Kinne, G. Malnic, and G. Giebisch. Renal bicarbonate reabsorption in the rat. I. Effects of hypokalemia and carbonic anhydrase. J. Clin. Invest. 78: 1558-1567, 1986[Medline].

2.   Chan, Y. L., G. Malnic, and G. Giebisch. Renal bicarbonate reabsorption in the rat. III. Distal tubule perfusion study of load dependence and bicarbonate permeability. J. Clin. Invest. 84: 931-938, 1989[Medline].

3.   Chu, T.-S., Y. Peng, A. Cano, M. Yanagisawa, and R. J. Alpern. EndothelinB receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells. J. Clin. Invest. 97: 1454-1462, 1996[Abstract/Free Full Text].

4.   Crayen, M. L., and W. Thoenes. Architecture and cell structures in the distal nephron of the rat kidney. Cytobiologie 17: 197-211, 1978[Medline].

5.   Eiam-Ong, S., S. A. Hilden, A. J. King, C. A. Johns, and N. E. Madias. Endothelin-1 stimulates the Na+/H+ and Na+/HCO<SUP>−</SUP><SUB>3</SUB> transporters in rabbit renal cortex. Kidney Int. 42: 18-24, 1992[Medline].

6.   Emmons, C., and J. B. Stokes. Cellular actions of cAMP on HCO<SUP>−</SUP><SUB>3</SUB>-secreting cells of rabbit CCD: dependence on in vivo acid-base status. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F528-F535, 1984.

7.   Garvin, J., and K. Sanders. Endothelin inhibits fluid and bicarbonate transport in part by reducing Na+/K+ ATPase activity in the rat proximal straight tubule. J. Am. Soc. Nephrol. 2: 976-982, 1991[Abstract].

8.   Hebert, R. L., L. Regnier, and L. N. Peterson. Rabbit cortical collecting ducts express a novel prostacyclin receptor. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F145-F154, 1995[Abstract/Free Full Text].

9.   Hirata, Y., H. Matsuoka, K. Kimura, K. Fukui, H. Hayakawa, E. Suzuki, T. Sugimoto, T. Sugimoto, M. Yanagisawa, and T. Masaki. Renal vasoconstriction by the endothelial cell-derived peptide endothelin in spontaneously hypertensive rats. Circ. Res. 65: 1370-1379, 1989[Abstract].

10.   Horie, S., O. Moe, A. Tehedor, and R. J. Alpern. Preincubation in acid medium increases Na/H antiporter activity in cultured renal proximal tubule cells. Proc. Natl. Acad. Sci. USA 87: 4742-4745, 1990[Abstract].

11.   Korbmacher, C., E. L. Boulpaep, G. Giebisch, and J. Geibel. Endothelin increases [Ca2+]i in M-1 mouse cortical collecting duct cells by a dual mechanism. Am J. Physiol. 265 (Cell Physiol. 34): C349-C357, 1993[Abstract/Free Full Text].

12.   Kriz, W., and B. Kaissling. Structural organization of the mammalian kidney. In: The Kidney. Physiology and Pathophysiology, edited by D. Seldin, and G. Giebisch. New York: Raven, 1992, p. 707-777.

13.   Levine, D. Z., M. Iacovitti, and V. Harrison. Bicarbonate secretion in vivo by rat distal tubules during alkalosis induced by dietary chloride restriction and alkali loading. J. Clin. Invest. 87: 1513-1518, 1991[Medline].

14.   Levine, D. Z., M. Iacovitti, L. Nash, and D. Vandorpe. Secretion of bicarbonate by rat distal tubules in vivo. Modulation by overnight fasting. J. Clin. Invest. 81: 1873-1878, 1988[Medline].

15.   Ling, B. N. Effect of luminal endothelin-1 on apical Na+ and Cl- channels in primary cultured rabbit CCT (Abstract). Clin. Res. 43: 47A, 1995.

16.   Ma, Y. H., H. P. Reusch, E. Wilson, J. A. Escobedo, W. J. Fantl, L. T. Williams, and H. E. Ives. Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma. J. Biol. Chem. 269: 30734-30739, 1994[Abstract/Free Full Text].

17.   Marsen, T. A., H. Schramek, and M. J. Dunn. Renal actions of endothelin: linking cellular signaling pathways to kidney disease. Kidney Int. 45: 336-344, 1994[Medline].

18.   Moncada, S. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2, and prostacyclin. Pharmacol. Rev. 30: 293-331, 1979[Medline].

19.   Roman, R. J., M. L. Kauker, N. A. Terragno, and P. Y. K. Wong. Inhibition of renal prostaglandin synthesis and metabolism by indomethacin in rats. Proc. Soc. Exp. Biol. Med. 159: 165-170, 1978.

20.   Sabatini, S. S., M. E. Laski, and N. A. Kurtzman. NEM-sensitive ATPase activity in the rat nephron: effect of metabolic acidosis and alkalosis. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F297-F307, 1990[Abstract/Free Full Text].

21.   Simonson, M. S., S. Wann, P. Mene, G. R. Dubyak, M. Kester, Y. Nakazato, J. R. Sedor, and M. J. Dunn. Endothelin stimulates phoshpholipase C, Na+/K+ exchange, c-fos expression and mitogenesis in rat mesangial cells. J. Clin. Invest. 83: 708-712, 1989[Medline].

22.   Star, R. A. Quantitation of total carbon dioxide in nanoliter samples by flow-through fluorometry. Am J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F429-F432, 1990[Abstract/Free Full Text].

23.   Terada, T., K. Tomita, H. Nonoguchi, and F. Marumo. Different localization of two types of endothelin receptor mRNA in microdissected rat nephron segments using reverse transcription and polymerase chain reaction assay. J. Clin. Invest. 90: 107-112, 1992[Medline].

24.   Tomita, K., H. Nonguchi, and F. Marumo. Effects of endothelin on peptide-dependent cyclic adenosine monophosphate accumulation along the nephron segments of the rat. J. Clin. Invest. 85: 2014-2018, 1990[Medline].

25.   Tomita, K., H. Nonoguchi, and F. Marumo. Regulation of NaCl transport by endothelin in renal tubules. Semin. Nephrol. 12: 30-36, 1992[Medline].

26.   Tsukahara, H., H. Ende, H. I. Magazine, W. F. Bahou, and M. S. Goligorsky. Molecular and functional characterization of the non-isopeptide-selective ETB receptor in endothelial cells. Receptor coupling to nitric oxide synthase. J. Biol. Chem. 269: 21778-21785, 1994[Abstract/Free Full Text].

27.   Wang, T., G. Malnic, G. Giebisch, and Y. L. Chan. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule. J. Clin. Invest. 91: 2776-2784, 1993[Medline].

28.   Weinman, E. J., S. Shenolikar, and A. M. Kahn. cAMP-associated inhibition of Na+-H+ exchanger in rabbit kidney brush border membranes. Am. J. Physiol. 252 (Renal Fluid Electrolyte Physiol. 21): F19-F25, 1987[Abstract/Free Full Text].

29.   Wesson, D. E. Depressed distal tubule acidification corrects chloride-deplete alkalosis in rats. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F636-F644, 1990[Abstract/Free Full Text].

30.   Wesson, D. E. Dietary HCO3 reduces distal tubule acidification by increasing cellular HCO3 secretion. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F132-F142, 1996[Abstract/Free Full Text].

31.   Wesson, D. E. Prostacyclin increases distal tubule HCO3 secretion in the rat. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F1183-F1192, 1996[Abstract/Free Full Text].

32.   Wesson, D. E. Endogenous endothelins mediate increased distal tubule acidification induced by dietary acid in rats. J. Clin. Invest. 99: 2203-2211, 1997[Abstract/Free Full Text].

33.   Wesson, D. E., and G. M. Dolson. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F308-F317, 1991[Abstract/Free Full Text].

34.   Wesson, D. E., J. Simoni, and D. F. Green. Acidic extracellular pH increases endothelin-1 secretion by human endothelial cells of glomerular but not aortic origin (Abstract). J. Invest. Med. 44: 336A, 1996.

35.   Wong, T. Regulation of HCO3 and Na+ transport by nitric oxide and cGMP in kidney proximal tubule (Abstract). J. Am. Soc. Nephrol. 6: 634, 1995.


AJP Renal Physiol 273(4):F586-F594
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