Na/H exchange and H-K ATPase increase distal tubule acidification in chronic alkalosis
1998; Elsevier BV; Volume: 53; Issue: 4 Linguagem: Inglês
10.1111/j.1523-1755.1998.00838.x
ISSN1523-1755
Autores Tópico(s)Aldose Reductase and Taurine
ResumoNa+/H+ exchange and H-K ATPase increase distal tubule acidification in chronic alkalosis. We examined whether H+-ATPase, H+-K+-ATPase, and or Na+/H+ exchange mediates increased distal tubule acidification in animals with chronic metabolic alkalosis using pharmacological inhibitors of these H+ transporters in in vivo-perfused tubules of anesthetized rats. Chronic metabolic alkalosis was induced with furosemide followed by minimum electrolyte diet and HCO3 drinking water. The reduction in net HCO3 reabsorption was greater in distal tubules of alkalotic compared to control animals perfused with Schering 28080 to inhibit H+-K+-ATPase (-6.4 ± 0.9 vs. -1.4 ± 0.5 pmol/mm · min-1, P < 0.02) and with EIPA to inhibit Na+/H+ exchange (-11.1 ± 1.7 vs. -6.6 ± 0.9 pmol/mm · min-1, P < 0.01) but was similar in distal tubules of alkalotic and control animals perfused with bafilomycin to inhibit H+-ATPase. The greater reduction of distal tubule net HCO3 reabsorption in alkalotic compared to control animals induced by EIPA was eliminated by systemic infusion of the endothelin receptor antagonist bosentan (-4.6 ± 0.7 vs. -4.4 ± 0.7 pmol/mm · min-1, P = NS) but the greater reduction induced by Schering 28080 persisted. Urine endothelin-1 (ET-1) excretion was higher in animals with maintained alkalosis (164.5 ± 23.7 vs. 76.6 ± 10.8 fmol/day, P < 0.03), but decreased following KCl repletion to a value (86.7 ± 10.0 fmol/day, P < 0.02 vs. respective before-KCl value) that was not different from that for KCl-repleted control animals (79.9 ± 8.7 fmol/day, P = NS vs. KCl-repleted alkalotic animals). The data support that augmented distal tubule acidification in alkalotic animals is due to increased H+-K+-ATPase and Na+/H+ exchange activity, the latter stimulated by endogenous endothelins. Na+/H+ exchange and H-K ATPase increase distal tubule acidification in chronic alkalosis. We examined whether H+-ATPase, H+-K+-ATPase, and or Na+/H+ exchange mediates increased distal tubule acidification in animals with chronic metabolic alkalosis using pharmacological inhibitors of these H+ transporters in in vivo-perfused tubules of anesthetized rats. Chronic metabolic alkalosis was induced with furosemide followed by minimum electrolyte diet and HCO3 drinking water. The reduction in net HCO3 reabsorption was greater in distal tubules of alkalotic compared to control animals perfused with Schering 28080 to inhibit H+-K+-ATPase (-6.4 ± 0.9 vs. -1.4 ± 0.5 pmol/mm · min-1, P < 0.02) and with EIPA to inhibit Na+/H+ exchange (-11.1 ± 1.7 vs. -6.6 ± 0.9 pmol/mm · min-1, P < 0.01) but was similar in distal tubules of alkalotic and control animals perfused with bafilomycin to inhibit H+-ATPase. The greater reduction of distal tubule net HCO3 reabsorption in alkalotic compared to control animals induced by EIPA was eliminated by systemic infusion of the endothelin receptor antagonist bosentan (-4.6 ± 0.7 vs. -4.4 ± 0.7 pmol/mm · min-1, P = NS) but the greater reduction induced by Schering 28080 persisted. Urine endothelin-1 (ET-1) excretion was higher in animals with maintained alkalosis (164.5 ± 23.7 vs. 76.6 ± 10.8 fmol/day, P < 0.03), but decreased following KCl repletion to a value (86.7 ± 10.0 fmol/day, P < 0.02 vs. respective before-KCl value) that was not different from that for KCl-repleted control animals (79.9 ± 8.7 fmol/day, P = NS vs. KCl-repleted alkalotic animals). The data support that augmented distal tubule acidification in alkalotic animals is due to increased H+-K+-ATPase and Na+/H+ exchange activity, the latter stimulated by endogenous endothelins. Chronic metabolic alkalosis is maintained in part by increased distal tubule acidification1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar,2.Wesson D.E. Depressed distal tubule acidification corrects chloride-deplete alkalosis in rats.Am J Physiol. 1990; 259 Renal Fluid Electrolyte Physiol 28: F636-F644Google Scholar due to augmented proton (H+) secretion in this nephron segment3.Wesson D.E. Dolson G.M. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis.Am J Physiol. 1991; 261 Renal Fluid Electrolyte Physiol 30: F308-F317Google Scholar,4.Wesson D.E. Dolson G.M. Maximal proton secretory rate of rat distal tubules is higher during chronic metabolic alkalosis.Am J Physiol. 1991; 261 (Renal Fluid Electrolyte Physiol 30): F753-F759PubMed Google Scholar, but the H+ transporters that meditate this increment in distal tubule acidification are not known. At least three H+ transporters contribute to luminal H+ secretion in the rat distal nephron segment accessible to micropuncture. Vacuolar H+-ATPase mediates H+ secretion in this segment of control rats5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar and its activity is increased by dietary acid6.Sabatini S.S. Laski M.E. Kurtzman N.A. NEM-sensitive ATPase activity in the rat nephron: Effect of metabolic acidosis and alkalosis.Am J Physiol. 1990; 258Renal Fluid Electrolyte Physiol 27: F297-F307Google Scholar,7.Eiam-Ong S. Kurztman N.A. Sabatini S. Regulation of collecting tubule adenosine triphosphatases by aldosteone and potassium.J Clin Invest. 1993; 91: 2385-2392Crossref PubMed Scopus (77) Google Scholar. H+-K+-ATPase contributes little to rat distal tubule H+ secretion in controls5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar but does so significantly in those with K+-depletion5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar,8.Cheval L. Bartlet-Bas C. Khadouri C. Feraille E. Marsy S. Doucet A. K+-ATPase-mediated Rb+ transport in rat collecting tubule: Modulation during K+ deprivation.Am J Physiol. 1991; 260 Renal Fluid Electrolyte Physiol 29: F800-F805Google Scholar, an etiologically important factor in some models of chronic alkalosis9.Wesson D.E. Combined K+ and Cl- repletion corrects augmented H+ secretion by distal tubules in chronic alkalosis.Am J Physiol. 1994; 266 (Renal Fluid Electrolyte Physiol 35): F592-F603Google Scholar. Na+/H+ exchange also mediates distal tubule luminal H+ secretion in controls5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar but the factors that modulate this process are less well described. Nonetheless, endothelin-1 increases Na+/H+ exchange activity renal epithelia in vitro10.Chu T.-S. Peng Y. Cano A. Yanagisawa M. Alpern R.J. EndothelinB receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells.J Clin Invest. 1996; 97: 1454-1462Crossref PubMed Scopus (67) Google Scholar, systemic infusion increases distal tubule acidification in vivo11.Wesson D.E. Dolson G.M. Endothelin-1 increases rat distal tubule acidification in vivo.Am J Physiol. 1997; 273 Renal Physiol 42: F586-F594Google Scholar, and endogenous endothelins mediate increased distal tubule acidification induced by dietary acid12.Wesson D.E. Endogenous endothelins mediate increased distal tubule acidification induced by dietary acid in rats.J Clin Invest. 1997; 99: 2203-2211Crossref PubMed Scopus (60) Google Scholar. The present studies investigated relative contributions of vacuolar H+-ATPase, H+-K+-ATPase, and Na+/H+ exchange to increased distal tubule acidification in rats with chronic metabolic alkalosis. These in vivo microperfusion studies show that pharmacological inhibitors of Na+/H+ exchange and of H+-K+-ATPase reduce the increment in distal tubule acidification between alkalotic and control animals but an H+-ATPase inhibitor did not. Furthermore, the increment in distal tubule acidification in alkalotic animals sensitive to inhibition of Na+/H+ exchange was eliminated by endothelin receptor inhibition. The data support that increased distal tubule acidification in alkalotic animals is due to increased H+-K+-ATPase and of Na+/H+ exchange activity, the latter stimulated by endogenous endothelins. Male and female Munich-Wistar rats (Harlan Sprague-Dawley, Houston, TX, USA) weighing 230 to 267 g and eating a minimum electrolyte diet (ICN Nutritional Biochemicals, Cleveland, OH, USA) were studied. Distal tubule net HCO3 reabsorption was measured using in vivo microperfusion in controls and animals with chronic metabolic alkalosis induced as described previously1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar. Briefly, all animals received three intraperitoneal injections of furosemide (10 mg/kg/dose) in 12 hour intervals while ingesting distilled H2O. Forty-eight hours after the first furosemide dose, drinking solution of alkalotic animals was changed to 40 mM NaHCO3+ 40 mM KHCO3 and that of control animals to 40 mM NaCl + KCl. Animals continued the low electrolyte diet and were studied four to five weeks after protocol initiation. Because endothelin-1 (ET-1) increases distal tubule acidification11.Wesson D.E. Dolson G.M. Endothelin-1 increases rat distal tubule acidification in vivo.Am J Physiol. 1997; 273 Renal Physiol 42: F586-F594Google Scholar and endogenous endothelins mediate augmented distal tubule acidification12.Wesson D.E. Endogenous endothelins mediate increased distal tubule acidification induced by dietary acid in rats.J Clin Invest. 1997; 99: 2203-2211Crossref PubMed Scopus (60) Google Scholar, some animals received the endothelinA and endothelinB receptor antagonist, Bosentan13.Clozel M. Breu V. Gray G. Kalina B. Loffler B.-M. Burri K. Cassal J.-M. Hirth G. Muller M. Neidhart W. Ramuz H. Pharmacological characterization of Bosentan, a new potent orally active nonpeptide endothelin receptor antagonist.J Pharmacol Exp Ther. 1994; 270: 228-235PubMed Google Scholar (Hoffman-LaRoche, Basel, Switzerland) 10 mg/kg i.v. 90 minutes before micropuncture to explore an endothelin role in the augmented distal tubule acidification of chronic metabolic alkalosis. This bosentan dose inhibits initial depressor and sustained pressor responses to ET-1 > 60% for at least six hours13.Clozel M. Breu V. Gray G. Kalina B. Loffler B.-M. Burri K. Cassal J.-M. Hirth G. Muller M. Neidhart W. Ramuz H. Pharmacological characterization of Bosentan, a new potent orally active nonpeptide endothelin receptor antagonist.J Pharmacol Exp Ther. 1994; 270: 228-235PubMed Google Scholar. Animals were prepared for micropuncture as previously described1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar. A minimum of four distal tubule segments were identified for microperfusion before using the animal for study. This nephron segment is comprised of multiple epithelia14.Crayen M.L. Thoenes W. Architecture and cell structures in the distal nephron of the rat kidney.Cytobiologie. 1978; 17: 197-211PubMed Google Scholar, hereafter referred to as “distal tubule” for simplicity. Tubules were microperfused during a 90 minute period beginning 30 minutes after kidney immobilization. Perfusion and collection pipettes were positioned in the earliest and latest (before tubule branching) portions, respectively, of accessible distal tubules. Perfusions were done at 6 nl/min, the early distal flow rate measured in free-flow studies2.Wesson D.E. Depressed distal tubule acidification corrects chloride-deplete alkalosis in rats.Am J Physiol. 1990; 259 Renal Fluid Electrolyte Physiol 28: F636-F644Google Scholar, calibrated in vitro and verified in vivo using the product of collected flow rate and the collected-to-perfusate inulin ratio. Only experiments in which these two flow rates were between 5 to 7 nl/min and within 10% of each other were accepted. Paired perfusions done with and without H+ transport inhibitors determined relative contributions of specific H+ transporters to distal tubule net HCO3 reabsorption. Because the one solution of the perfusion pair containing the H+ transport inhibitor was always perfused last, paired perfusions were done with identical solutions without H+ transport inhibitors as time controls. An injected latex cast determined perfused tubule length after acid digestion of the kidney1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar. Arterial blood (0.35 ml) was obtained anaerobically before micropuncture and plasma analyzed for tCO2 using microcalorimetry1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar or flow-through fluorometry15.Star R.A. Quantitation of total carbon dioxide in nanoliter samples by flow-through fluorometry.Am J Physiol. 1990; 258 Renal Fluid Electrolyte Physiol 27: F429-F432Google Scholar as described below and for pH, PCO2, and electrolytes as described previously1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar. Stellate vessel blood was obtained16.Dubose JR, T.D. Pucacco L.R. Seldin D.W. Carter N.W. Kokko J.P. Direct determination of PCO2 in the rat renal cortex.J Clin Invest. 1978; 62: 338-348Crossref PubMed Scopus (33) Google Scholar for total CO2 (tCO2) measurement as done previously3.Wesson D.E. Dolson G.M. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis.Am J Physiol. 1991; 261 Renal Fluid Electrolyte Physiol 30: F308-F317Google Scholar. Diet, but not drinking solution, was withheld the evening before studying micropunctured animals, yielding higher baseline HCO3 reabsorption17.Levine D.Z. Iacovitti M. Nash L. Vandorpe D. Secretion of bicarbonate by rat distal tubules in vivo. Modulation by overnight fasting.J Clin Invest. 1988; 81: 1873-1878Crossref PubMed Scopus (30) Google Scholar and permitting differences in HCO3 reabsorption to be more clearly seen. Table 1 shows that baseline perfusate [HCO3] was 7.5 mM, providing distal tubule HCO3 delivery that was comparable to that for alkalotic animals when perfused at 6 nl/min2.Wesson D.E. Depressed distal tubule acidification corrects chloride-deplete alkalosis in rats.Am J Physiol. 1990; 259 Renal Fluid Electrolyte Physiol 28: F636-F644Google Scholar. The perfusate was Cl--free to minimize HCO3 secretion and thereby permit more focused study of H+ secretion3.Wesson D.E. Dolson G.M. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis.Am J Physiol. 1991; 261 Renal Fluid Electrolyte Physiol 30: F308-F317Google Scholar. Bafilomycin (10-7M) was added to one solution of a perfusion pair to inhibit H+ ATPase in this nephron segment with high specificity18.Bowman E.J. Siebers A. Altendorf K. Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells.Biochem. 1988; 85: 7972-7976Google Scholar to determine the contribution of this H+ transporter to distal tubule net HCO3 reabsorption. Schering compound 28080 (Sch 28080) was added (10-5M) to inhibit H+-K+-ATPase8.Cheval L. Bartlet-Bas C. Khadouri C. Feraille E. Marsy S. Doucet A. K+-ATPase-mediated Rb+ transport in rat collecting tubule: Modulation during K+ deprivation.Am J Physiol. 1991; 260 Renal Fluid Electrolyte Physiol 29: F800-F805Google Scholar and determine its contribution to distal tubule net HCO3 reabsorption5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar. Sch 28080 decreases H+-ATPase activity in turtle urinary bladders when applied at doses higher than those used in the present studies19.Kohn O. Mitchell P.P. Steinmetz P.R. Sch-28080 inhibits bafilomycin-sensitive H+ secretion in turtle bladder independently of luminal [K+].Am J Physiol. 1993; 265 Renal Fluid Electrolyte Physiol 34: F174-F179Google Scholar, but does not affect H+-ATPase activity in rat cortical collecting ducts8.Cheval L. Bartlet-Bas C. Khadouri C. Feraille E. Marsy S. Doucet A. K+-ATPase-mediated Rb+ transport in rat collecting tubule: Modulation during K+ deprivation.Am J Physiol. 1991; 260 Renal Fluid Electrolyte Physiol 29: F800-F805Google Scholar. Ethyl-isopropylamiloride (EIPA) at 10-5M assessed the contribution of Na+/H+ exchange to HCO3 reabsorption in this segment5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar. The parent compound amiloride inhibits rat cortical collecting tubule H+-ATPase activity in vitro when added at doses higher than those used in the present studies, but does not inhibit H+-ATPase activity at doses comparable to those used in the present studies20.Dafnis E. Kurtzman N.A. Sabatini S. Effect of lithium and amiloride on collecting tubule transport enzymes.J Phar Exp Ther. 1992; 261: 701-706PubMed Google Scholar. Finally, amiloride does not affect rat cortical collecting duct H+-K+-ATPase activity even at high doses21.EIAM>-Ong S. Dafnis E. Spohn M. Kurtzman N.A. Sabatini S. H-K-ATPase in distal renal tubular acidosis: Urinary tract obstruction, lithium, and amiloride.Am J Physiol. 1993; 265 Renal Fluid Electrolyte Physiol 34: F875-F880Google Scholar.Table 1Perfusate composition Immediately after the experiment termination, samples of initial and collected perfusate as well as stellate vessel plasma were analyzed for inulin as done previously3.Wesson D.E. Dolson G.M. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis.Am J Physiol. 1991; 261 Renal Fluid Electrolyte Physiol 30: F308-F317Google Scholar. tCO2 was measured using microcalorimetry3.Wesson D.E. Dolson G.M. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis.Am J Physiol. 1991; 261 Renal Fluid Electrolyte Physiol 30: F308-F317Google Scholar in samples from alkalotic and control animals not given bosentan, and flow-through ultrafluorometry22.Wesson D.E. Dietary HCO3 reduces distal tubule acidification by increasing cellular HCO3 secretion.Am J Physiol. 1996; 271Renal Fluid Electrolyte Physiol 40: F132-F142Google Scholar was used in bosentan-treated alkalotic and control animals. For each tCO2 measuring system, determinations were done on the experimental day by comparing peak area of a 7 to 8 nl sample aliquot (corrected for that of a distilled H2O blank run concomitantly with each sample group) to that of a standard constructed for each sample run using an identical volume of the following NaHCO3 standards: 0, 2.5, 5, 10, 25, and 50 mM. A 5 mM NaHCO3 standard sample run five consecutive times yielded 4.9 ± 0.2 mM with microcalorimetry and 5.1 ± 0.1 with ultrafluorometry. Urine ET-1 excretion was determined in a 24 hour collection on day 28 of the induction protocol for alkalotic and control animals. Urine [ET-1] was measured with a RIA kit (Peninsula Laboratories, Inc., Belmont, CA, USA) after disposable column extraction (Sep-Pak C18, Milford, MA, USA) preconditioned with methanol, H2O, and acetic acid as described23.Benigni A. Perico N. Gaspari F. Zoja C. Bellizzi L. Gabanelli M. Remuzzi G. Increased renal endothelin production in rats with reduced renal mass.Am J Physiol. 1991; 260Renal Fluid Electrolyte Physiol 29: F331-F339Google Scholar and done in this laboratory12.Wesson D.E. Endogenous endothelins mediate increased distal tubule acidification induced by dietary acid in rats.J Clin Invest. 1997; 99: 2203-2211Crossref PubMed Scopus (60) Google Scholar. In vitro ET-1 recoveries for rat urine spiked with [125I]-ET-1 (ICM Biomedicals, Irvine, CA, USA) was 71 ± 2%. Because aldosterone increases rat cortical collecting duct H+-ATPase activity7.Eiam-Ong S. Kurztman N.A. Sabatini S. Regulation of collecting tubule adenosine triphosphatases by aldosteone and potassium.J Clin Invest. 1993; 91: 2385-2392Crossref PubMed Scopus (77) Google Scholar and thereby might mediate augmented distal tubule acidification in alkalotic animals, plasma aldosterone levels were measured four weeks after beginning respective protocols in each of four alkalotic and control animals by radioimmunoassay (COAT-A-COUNT®; DPC, Los Angeles, CA, USA). Bafilomycin A1 was obtained from WAKO Chemicals (Richmond, VA, USA) and Sch 28080 from Schering Plough (Kenilworth, NJ, USA). All other compounds were from Sigma Chemicals (St. Louis, MO, USA). Net HCO3 transport was the difference between perfused and collected rates, recognizing that a small amount of HCO3 secretion occurs even with Cl--free perfusates3.Wesson D.E. Dolson G.M. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis.Am J Physiol. 1991; 261 Renal Fluid Electrolyte Physiol 30: F308-F317Google Scholar. The decrease in net HCO3 reabsorption induced by a H+ transport inhibitor was compared between alkalotic and control animals to compare the contribution of the inhibited transporter to distal tubule acidification between groups. Each inhibitor was assumed to be specific for the intended transporter, recognizing that specificity might be < 100% as discussed. One distal tubule was perfused successfully for each perturbation per animal so that “N” represents both one tubule and one animal. 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 alkalotic animals. Table 2 compares plasma electrolyte and acid-base composition between groups. Alkalotic animals had higher tCO2 in both systemic and stellate vessel plasma and higher pH in systemic arterial plasma. By contrast, alkalotic animals had lower values for plasma [K+] and [Cl-].Table 2Plasma composition of micropunctured animals Tables 3 to Table 6 show that distal tubule net HCO3 reabsorption in did not change between paired perfusions in time controls of any group. Table 3 shows that bafilomycin and EIPA decreased net HCO3 reabsorption in distal tubules of control animals but net HCO3 reabsorption was not different in distal tubules perfused with Sch 28080. By contrast, Table 4 shows that bafilomycin, Sch 28080, and EIPA all decreased net HCO3 reabsorption in distal tubules of alkalotic animals. Similarly, Table 5 shows that bafilomycin and EIPA but not Sch 28080 decreased distal tubule net HCO3 reabsorption in bosentan-treated control animals and Table 6 shows that all three H+ transport inhibitors decreased net HCO3 reabsorption in distal tubules of bosentan-treated alkalotic animals.Table 3HCO3 reabsorption by distal tubules of control animals pair-perfused with solution #1 without and with the indicated H+ transport inhibitorTable 6Bicarbonate reabsorption by distal tubules of bosentan-treated animals with chronic metabolic alkalosis pair-perfused with solution #1 without and with the indicated H+ transport inhibitorTable 6Bicarbonate reabsorption by distal tubules of bosentan-treated animals with chronic metabolic alkalosis pair-perfused with solution #1 without and with the indicated H+ transport inhibitorTable 4HCO3 reabsorption by distal tubules of animals with chronic metabolic alkalosis pair-perfused with solution #1 without and with the indicated H+ transport inhibitorTable 4HCO3 reabsorption by distal tubules of animals with chronic metabolic alkalosis pair-perfused with solution #1 without and with the indicated H+ transport inhibitorTable 5Bicarbonate reabsorption by distal tubules of bosentan-treated control animals pair-perfused with solution #1 without and with the indicated H+ transport inhibitorTable 5Bicarbonate reabsorption by distal tubules of bosentan-treated control animals pair-perfused with solution #1 without and with the indicated H+ transport inhibitor Figure 1 shows the change in distal tubule HCO3 reabsorption between the second and first perfusions of the perfusion pairs in animals not given bosentan. The change in net HCO3 reabsorption between the second and first perfusion in all time controls was not different from zero in all groups. By contrast, the change in distal tubule HCO3 reabsorption was significantly less than zero and significantly less than its respective time control in alkalotic and control animals perfused with bafilomycin and EIPA and in alkalotic animals perfused with Sch 28080. The decrease in HCO3 reabsorption was greater in distal tubules of alkalotic compared to control animals perfused with Sch 28080 (-6.4 ± 0.9 vs. -1.4 ± 0.5 pmol/mm · min-1, P < 0.02) and EIPA (-11.1 ± 1.7 vs. -6.6 ± 0.9 pmol/mm · min-1, P < 0.01), but the decrease in HCO3 reabsorption was similar in distal tubules of alkalotic and control animals perfused with bafilomycin (-6.3 ± 1.0 vs. -4.2 ± 0.7 pmol/mm · min-1, P = NS). Identical comparisons for bosentan-treated alkalotic and control animals are depicted in Figure 2. As was seen in animals not given bosentan, the decrease in HCO3 reabsorption was greater in distal tubules of bosentan-treated alkalotic compared to control animals perfused with Sch 28080 (-6.1 ± 0.9 vs. -1.8 ± 0.7 pmol/mm · min-1, P < 0.02) and was similar in alkalotic and control animals perfused with bafilomycin (-3.3 ± 0.6 vs. -3.9 ± 0.6 pmol/mm · min-1, P = NS). In contrast to the animals not given bosentan, Figure 2 shows that the decrease in HCO3 reabsorption was similar in distal tubules of bosentan-treated alkalotic and control animals perfused with EIPA (-4.6 ± 0.7 vs. -4.4 ± 0.7 pmol/mm · min-1, P = NS).Figure 2Change in distal tubule net HCO3reabsorption between the last and first perfusions of perfusion pairs in animals given bosentan. Abbreviations are: Baf, bafilomycin (10-7M); Sch, Schering 28080 (10-5M); and EIPA, ethyl-isopropylamiloride (10-5M). Data are means ±SE. *P < 0.05 versus respective time control. +P < 0.05 versus respective control group. Symbols are: (□) control; () alkalosis.View Large Image Figure ViewerDownload (PPT) The bosentan data showing amelioration of augmented EIPA-sensitive distal tubule acidification in alkalotic animals by this endothelin receptor antagonist are consistent with an endothelin role in mediating increased distal tubule acidification in alkalotic animals. Because K+ repletion corrects augmented distal tubule acidification in alkalotic animals9.Wesson D.E. Combined K+ and Cl- repletion corrects augmented H+ secretion by distal tubules in chronic alkalosis.Am J Physiol. 1994; 266 (Renal Fluid Electrolyte Physiol 35): F592-F603Google Scholar, plasma tCO2 concentration and urine ET-1 excretion were measured in four each of alkalotic and control animals before and 24 hours after beginning 80 mM KCl drinking solution. Alkalotic and control animals had respective tail vein plasma tCO2 concentrations of 32.9 ± 1.9 and 25.1 ± 1.5 mM four weeks into their respective induction protocols and were 26.7 ± 1.7 and 24.8 ± 1.5 mM, respectively, 24 hours following initiation of the KCl drinking solution. Figure 3 shows that urine ET-1 excretion was higher in alkalotic compared to control animals before KCl (164.5 ± 23.7 vs. 76.6 ± 10.8 fmol/day, P < 0.03) but decreased following KCl repletion in alkalotic animals to a value (86.7 ± 10.0 fmol/day, P < 0.02 vs. respective before-KCl value) not different from that for KCl-repleted control animals (79.9 ± 8.7 fmol/day, P = NS vs. KCl-repleted alkalotic animals). Plasma aldosterone concentration was lower in alkalotic compared to control animals (1.5 ± 0.2 vs. 3.8 ± 0.4 ng/dl, P < 0.002). The kidney helps to maintain some types of chronic metabolic alkalosis by excreting acid despite extracellular fluid alkalosis1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar. In vivo microperfusion studies in rats with chronic metabolic alkalosis show that increased distal tubule acidification1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar,2.Wesson D.E. Depressed distal tubule acidification corrects chloride-deplete alkalosis in rats.Am J Physiol. 1990; 259 Renal Fluid Electrolyte Physiol 28: F636-F644Google Scholar mediated by increased H+ secretion3.Wesson D.E. Dolson G.M. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis.Am J Physiol. 1991; 261 Renal Fluid Electrolyte Physiol 30: F308-F317Google Scholar,4.Wesson D.E. Dolson G.M. Maximal proton secretory rate of rat distal tubules is higher during chronic metabolic alkalosis.Am J Physiol. 1991; 261 (Renal Fluid Electrolyte Physiol 30): F753-F759PubMed Google Scholar contributes to urine acid excretion in this metabolic disorder. Furthermore, K+ depletion in some models of chronic metabolic alkalosis1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar,9.Wesson D.E. Combined K+ and Cl- repletion corrects augmented H+ secretion by distal tubules in chronic alkalosis.Am J Physiol. 1994; 266 (Renal Fluid Electrolyte Physiol 35): F592-F603Google Scholar contributes to the augmented distal tubule acidification in these animals by mechanisms that remain poorly understood. The present studies show that inhibitors of H+-K+-ATPase and of Na+/H+ exchange reduce the increment distal nephron acidification between alkalotic and control animals, but an H+-ATPase inhibitor does not. Furthermore, endothelin receptor inhibition eliminates the increment in distal tubule acidification mediated by Na+/H+ exchange, consistent with an endothelin role in mediating augmented acidification. The data support that augmented distal tubule acidification in animals with chronic metabolic alkalosis is mediated by increased H+-K+-ATPase and increased Na+/H+ exchange activity, the latter stimulated by endogenous endothelins. Clarification of the mechanisms by which chronic metabolic alkalosis is maintained has been hampered by attempts to reconcile data from studies using different experimental preparations, techniques, and models of this metabolic disorder1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar. The present in vivo studies have the advantage of permitting tubule mechanisms to be assessed in situ but pose the challenge of defining the environmental factors that induce the altered tubule function. Second, pharmacologic inhibition of H+ transporters as done in the present studies allows in vivo assessment of acidification in a fashion that minimally disturbs overall tubule function, but the conclusions reached regarding the components of tubule acidification are valid only to the extent that the inhibitors used are specific for the transporters they are purported to inhibit. Nevertheless, specificity of the inhibitors used at the doses employed in the present studies appear sufficient to support the stated conclusions, as discussed in the Methods section. Third, data discussed apply to the present model and possibly to others with similar characteristics but extrapolating these findings to other models should be done with caution. The present model is K+ and Cl- deplete, excretes an acid urine, and has augmented acidification in both proximal and distal tubules1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar. Augmented distal tubule acidification in this model is mediated by increased H+ secretion3.Wesson D.E. Dolson G.M. Augmented bidirectional HCO3 transport by rat distal tubules in chronic alkalosis.Am J Physiol. 1991; 261 Renal Fluid Electrolyte Physiol 30: F308-F317Google Scholar,4.Wesson D.E. Dolson G.M. Maximal proton secretory rate of rat distal tubules is higher during chronic metabolic alkalosis.Am J Physiol. 1991; 261 (Renal Fluid Electrolyte Physiol 30): F753-F759PubMed Google Scholar, which is normalized by KCl but not by NaCl repletion9.Wesson D.E. Combined K+ and Cl- repletion corrects augmented H+ secretion by distal tubules in chronic alkalosis.Am J Physiol. 1994; 266 (Renal Fluid Electrolyte Physiol 35): F592-F603Google Scholar. It follows that K+ depletion, which is associated with stimulated distal tubule HCO3 reabsorption24.Capasso G. Kinne R. Malnic G. Giebisch G. Renal bicarbonate reabsorption in the rat. I. Effects of hypokalemia and carbonic anhydrase.J Clin Invest. 1986; 78: 1558-1567Crossref PubMed Scopus (40) Google Scholar, contributes importantly to increased distal tubule acidification in this model. Therefore, these findings more likely apply to models of chronic metabolic alkalosis with K+ depletion and less likely to those without it. The present studies show that Sch 28080 decreases distal tubule net HCO3 reabsorption in alkalotic but not control animals, supporting the hypothesis that increased H+-K+-ATPase activity contributes to the augmented distal tubule acidification in alkalotic animals. This H+-K+-ATPase inhibitor has little effect on distal tubule acidification in control rats5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar but K+ depletion induces expression of a Sch 28080-sensitive H+-K+-ATPase in the rat cortical collecting tubule25.Buffin-Meyer B. Younes-Ibrahim M. Barlet-Bas C. Cheval L. Marsy S. Doucet A. K depletion modifies the properties of Sch-28080-sensitive K-ATPase in rat collecting duct.Am J Physiol. 1997; 272 (Renal Physiol 41): F124-F131PubMed Google Scholar, an epithelium comprising the rat distal tubule accessible to micropuncture14.Crayen M.L. Thoenes W. Architecture and cell structures in the distal nephron of the rat kidney.Cytobiologie. 1978; 17: 197-211PubMed Google Scholar. K+ depletion is a component of the chronic alkalosis model used in the present studies1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar and K+ repletion restores augmented distal tubule acidification to a level comparable to control9.Wesson D.E. Combined K+ and Cl- repletion corrects augmented H+ secretion by distal tubules in chronic alkalosis.Am J Physiol. 1994; 266 (Renal Fluid Electrolyte Physiol 35): F592-F603Google Scholar. Although high Sch 28080 concentrations inhibit H+-ATPase activity in turtle bladders in vitro19.Kohn O. Mitchell P.P. Steinmetz P.R. Sch-28080 inhibits bafilomycin-sensitive H+ secretion in turtle bladder independently of luminal [K+].Am J Physiol. 1993; 265 Renal Fluid Electrolyte Physiol 34: F174-F179Google Scholar, even high concentrations do not affect activity of this transporter in the rat cortical collecting duct8.Cheval L. Bartlet-Bas C. Khadouri C. Feraille E. Marsy S. Doucet A. K+-ATPase-mediated Rb+ transport in rat collecting tubule: Modulation during K+ deprivation.Am J Physiol. 1991; 260 Renal Fluid Electrolyte Physiol 29: F800-F805Google Scholar. Furthermore, bafilomycin, a more specific H+-ATPase inhibitor18.Bowman E.J. Siebers A. Altendorf K. Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells.Biochem. 1988; 85: 7972-7976Google Scholar, clearly inhibits distal tubule acidification in both alkalotic and control animals whereas the effect of Sch 28080 was demonstrable in only alkalotic animals. Thus, the present studies clearly distinguish the effects of these two H+ transport inhibitors and support that part of the augmented distal tubule acidification in alkalotic animals is mediated by the described H+-K+-ATPase. The rat cortical collecting tubule has H+-ATPase activity7.Eiam-Ong S. Kurztman N.A. Sabatini S. Regulation of collecting tubule adenosine triphosphatases by aldosteone and potassium.J Clin Invest. 1993; 91: 2385-2392Crossref PubMed Scopus (77) Google Scholar and this transporter contributes to in vivo distal tubule acidification in control animals5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar. The H+-ATPase inhibitor bafilomycin decreased distal tubule acidification to a similar degree in alkalotic and control animals, supporting that activity of this transporter is not increased in the present model of chronic metabolic alkalosis. Systemic factors shown to increase cortical collecting tubule H+-ATPase activity include acid-loading6.Sabatini S.S. Laski M.E. Kurtzman N.A. NEM-sensitive ATPase activity in the rat nephron: Effect of metabolic acidosis and alkalosis.Am J Physiol. 1990; 258Renal Fluid Electrolyte Physiol 27: F297-F307Google Scholar,7.Eiam-Ong S. Kurztman N.A. Sabatini S. Regulation of collecting tubule adenosine triphosphatases by aldosteone and potassium.J Clin Invest. 1993; 91: 2385-2392Crossref PubMed Scopus (77) Google Scholar, aldosterone7.Eiam-Ong S. Kurztman N.A. Sabatini S. Regulation of collecting tubule adenosine triphosphatases by aldosteone and potassium.J Clin Invest. 1993; 91: 2385-2392Crossref PubMed Scopus (77) Google Scholar, and angiotensin II26.Tojo A. Tisher C.C. Madsen K.M. Angiotensin II regulates H+-ATPase activity in rat cortical collecting duct.Am J Physiol. 1994; 267 (Renal Fluid Electrolyte Physiol 36): F1045-F1051PubMed Google Scholar. The alkalotic animals of the present studies were not acid-loaded and their plasma aldosterone level was low, possibly due to K+ depletion27.Boyd J. Palmore W. Mulrow P.J. Role of potassium in the control of aldosterone secretion in the rat.Endocrinol. 1971; 88: 556-565Crossref PubMed Scopus (84) Google Scholar in this model of chronic alkalosis1.Wesson D.E. Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.J Clin Invest. 1989; 84: 1460-1469Crossref PubMed Scopus (27) Google Scholar. Lower plasma aldosterone might yield low H+-ATPase activity7.Eiam-Ong S. Kurztman N.A. Sabatini S. Regulation of collecting tubule adenosine triphosphatases by aldosteone and potassium.J Clin Invest. 1993; 91: 2385-2392Crossref PubMed Scopus (77) Google Scholar rather than the level comparable to control shown in the present studies. Whether some other phenomena also stimulates this transporter, counterbalancing the effect of low aldosterone levels, or whether the effect of aldosterone on this transporter differs in the present experimental model is unclear. Unpublished studies in our laboratory show that i.v. infusion of the angiotensin converting enzyme inhibitor captopril causes hypotension in the alkalotic animals, precluding distal tubule perfusion. The data are consistent with increased angiotensin II activity in the alkalotic animals, but whether this agent contributes to augmented acidification in this model of chronic alkalosis could not be determined. Na+/H+ exchange contributes to distal tubule acidification5.Wang T. Malnic G. Giebisch G. Chan Y.L. Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.J Clin Invest. 1993; 91: 2776-2784Crossref PubMed Scopus (71) Google Scholar and the larger EIPA-sensitive component of acidification in alkalotic animals suggests that increased Na+/H+ exchange mediates a portion of the augmented acidification in alkalotic animals. EIPA reduced distal tubule net HCO3 reabsorption without affecting tubule voltage as previously shown for hexamethylene-amiloride28.Fernandez R. Lopes M.J. De Lira R.F. Dantas W.F.G. Cragoe JR, E.J. Malnic G. Mechanism of acidfication along cortical distal tubule of the rat.Am J Physiol. 1994; 266 (Renal Fluid Electrolyte Physiol 35): F218-F220PubMed Google Scholar. The absence of an effect on tubule voltage reflects high specificity of these compounds for Na+/H+ exchange and low specificity for the Na+ channel29.Frelin C. Barbry P. Vigne P. Chassande O. Cragoe E.J. Lazdunski M. Amiloride and its analogs as tools to inhibit Na transport vial the Na channel, the Na/H antiport and the Na/Ca exchanger.Biochimie. 1988; 70: 1285-1290Crossref PubMed Scopus (83) Google Scholar. The present studies are consistent with a role for endogenous endothelins as mediators of augmented EIPA-sensitive distal tubule acidification in the alkalotic animals. Endothelin-1 (ET-1) increases Na+/H+ exchange in renal epithelia in vitro10.Chu T.-S. Peng Y. Cano A. Yanagisawa M. Alpern R.J. EndothelinB receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells.J Clin Invest. 1996; 97: 1454-1462Crossref PubMed Scopus (67) Google Scholar, increases distal tubule acidification when infused systemically11.Wesson D.E. Dolson G.M. Endothelin-1 increases rat distal tubule acidification in vivo.Am J Physiol. 1997; 273 Renal Physiol 42: F586-F594Google Scholar, and endogenous endothelins mediate increased distal tubule acidification induced by dietary acid12.Wesson D.E. Endogenous endothelins mediate increased distal tubule acidification induced by dietary acid in rats.J Clin Invest. 1997; 99: 2203-2211Crossref PubMed Scopus (60) Google Scholar. The present studies show that the high urine ET-1 excretion in animals with maintained alkalosis decreases to a level comparable to control (as did plasma tCO2) after KCl repletion. Previous studies from this laboratory show that KCl repletion normalizes augmented H+ secretion in the distal tubule of this model of chronic metabolic alkalosis9.Wesson D.E. Combined K+ and Cl- repletion corrects augmented H+ secretion by distal tubules in chronic alkalosis.Am J Physiol. 1994; 266 (Renal Fluid Electrolyte Physiol 35): F592-F603Google Scholar. The present studies support a model in which K+ depletion, a key determinant of chronic metabolic alkalosis, increases renal endothelin production which in turn increases distal tubule acidification. Confirmation of this hypothetical model awaits further studies. In summary, the present studies show that inhibitors of H+-K+-ATPase and of Na+/H+ exchange reduce the increment in distal tubule acidification between alkalotic and control animals but an H+-ATPase inhibitor does not. Endothelin receptor inhibition eliminated the increment in distal tubule acidification in alkalotic animals sensitive to inhibition of Na+/H+ exchange. Furthermore, the data show increased urine ET-1 excretion in animals with maintained alkalosis that was reduced to a level comparable to that in control animals in response to KCl repletion. The data support that increased distal tubule acidification in alkalotic animals is mediated by increased H+-K+-ATPase and increased Na+/H+ exchange activity, the latter stimulated by endogenous endothelins. This work was supported by funds from the Merit Review Program of the Department of Veterans Affairs, from NIH grant 5-RO1-DK 36199 to 10 (N.A. Kurtzman, P.I.), and from the Texas Tech University Health Sciences Center. I am grateful to Mr. Ernest Pace, Mrs. Cathy Hudson, and Ms. Geri Tasby for technical assistance, to Mr. Edward McGuire for animal care, and to Neil A. Kurtzman for continued support. I am also grateful to Martine Clozel M.D. for generously providing bosentan without which these studies would not have been possible.
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