Regulation of Acid-Base Transport in the Rat Thick Ascending Limb

Regulation of Acid-Base Transport in the Rat Thick Ascending Limb

Regulation of Acid-Base Transport in the Rat Thick Ascending Limb David W. Good, PhD • The thick ascending limb of the rat influences urinary net acid...

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Regulation of Acid-Base Transport in the Rat Thick Ascending Limb David W. Good, PhD • The thick ascending limb of the rat influences urinary net acid excretion by reabsorbing both bicarbonate and ammonium. The bicarbonate absorption is mediated predominantly by apical membrane Na+ -H+ exchange and occurs at rates that are comparable to or greater than rates measured in cortical and medullary collecting ducts. The ammonium absorption is mediated predominantly by apical membrane Na +-NH4 +-2CI- cotransport and enhances urinary ammonium excretion by promoting countercurrent multiplication of ammonium, which facilitates ammonium secretion Into medullary collecting ducts. Studies with medullary thick ascending limbs (MTAL) In vitro have shown that the regulation of these transport processes involves both acute responses to changes in the luminal and peritubular environment and adaptive changes in tubule transport capacity in response to chronic systemic acid-base perturbations. In particular, an increase in potassium concentration inhibits ammonium absorption with no effect on net bicarbonate absorption whereas vasopressin inhibits bicarbonate absorption with no effect on net ammonium absorption. Chronic metabolic acidosis causes an adaptive increase in the ability of the MTAL to reabsorb both bicarbonate and ammonium. These results demonstrate that the MTAL is a site of regulation of renal acid-base transport and that ammonium and bicarbonate transport rates can vary independently In this nephron segment. © 1989 by the National Kidney Foundation, Inc. INDEX WORDS: Thick ascending limb; urinary acidification; bicarbonate; ammonium; potassium; vasopressin; cyclic AMP; metabolic acidosis.

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N THE PAST, acid transport in the kidney and the control of urinary acidification were explained almost entirely on the basis of transport events occurring in segments of the proximal tubule and collecting duct. More recently, however, using the isolated, perfused tubule technique, we have shown that the thick ascending limb of Henle's loop also can contribute to urinary net acid excretion. The purpose of this article is to describe briefly some of the acid-base transport processes that are present in the thick ascending limb of the rat and to discuss factors that are involved in regulating acid-base transport in this nephron segment. TRANSPORT EVENTS AND MECHANISMS

The thick ascending limb performs at least two transport functions that influence urinary net acid excretion: (1) reabsorption of bicarbonate, and (2) reabsorption of ammonium. These processes are considered separately below. From the Renal Electrolyte Physiology Laboratory, Department of Medicine. University of Texas Medical Branch. Galveston. Supported by Grants No. DK38217 and DK01745 from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health and a grant-in-aid from the American Heart Association. Address reprint requests to David W. Good. PhD. Room 4.200 John Sealy Hospital E-62. University of Texas Medical Branch. Galveston. TX 77550. © 1989 by the National Kidney Foundation. Inc. 0272-6386/89/1404-0005$3.00/0

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Bicarbonate Absorption

Medullary and cortical thick ascending limbs from rats absorb bicarbonate when perfused in vitro with equal concentrations of HC03- in perfusate and bath. 1.2 The average rate of absorption under different experimental conditions ranges from 8 to 17 pmollmin/mm tubule length.l-s These rates are comparable with rates measured in vitro in cortical and medullary collecting ducts6-l1 and are sufficient to account for much of the 10 % to 15 % of filtered bicarbonate that is reabsorbed between the late proximal convoluted and early distal tubule of the rat in vivo.! Bicarbonate absorption by the thick ascending limb also can explain the finding in rats that luminal bicarbonate concentration and pH in the early distal tubule are much less than values measured at the bend of Henle's loop. 12,13 It is likely, therefore, that the thick ascending limb is an important site of bicarbonate absorption and urinary acidification in the rat. The cellular mechanisms involved in thick ascending limb bicarbonate absorption have been investigated to a limited extent. The rat thick ascending limb contains carbonic anhydrase and inhibition of this enzyme with acetazolamide markedly inhibits HC0 3- absorption. 2 The HC0 3- absorption occurs against both electrical and chemical gradients and therefore is an active process.1.2 The active HC0 3- absorption is dependent on the presence of sodium and transepithelial sodium transport and is inhibited by high

American Journal of Kidney Diseases. Vol XlV, No 4 (October). 1989: pp 262-266

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concentrations of luminal amiloride, consistent with mediation of the HC0 3- absorption by apical membrane Na +-H+ exchange. 2 The basolateral membrane of the rat cortical thick ascending limb contains an electrogenic Na/HC0 3 cotransporter!4 through which bicarbonate may exit the cell to complete its transcellular absorption. These transport mechanisms are similar to those involved in transcellular HC0 3- absorption in the renal proximal tubule!5 and amphibian diluting segment.!6 Ammonium Absorption

A second means by which the thick ascending limb influences urinary net acid excretion is by reabsorbing ammonium. Micropuncture studies in rats have shown that approximately 50% of the ammonium secreted by the proximal convoluted tubule is subsequently reabsorbed as the tubule fluid flows through the loop of Henle. 13,!7 Studies with isolated, perfused medullary and cortical thick ascending limbs have shown that these segments absorb ammonium in vitro at rates sufficient to account for ammonium absorbed along the loop segment of the rat in vivo.! Ammonium absorption by the thick ascending limb enhances urinary ammonium excretion by promoting countercurrent multiplication of ammonium in the renal medulla and causing ammonium to accumulate to high concentrations in the medullary interstitial fluid.1.18,!9 The high medullary ammonium concentrations then facilitate secretion of ammonium into the medullary collecting ducts. 1.18-20 Recent studies indicate that conditions such as chronic metabolic acidosis 5,20 and changes in systemic potassium balance 2 1.22 may influence urinary ammonium excretion, in part, by affecting ammonium absorption in the medullary thick ascending limb and altering countercurrent multiplication of ammonium in the renal medulla (see below). Studies of the mechanism of ammonium transport in thick ascending limbs have revealed that the ammonium absorption is due to the direct transport of ammonium ions (NH4 +). This conclusion was based on the observation that, because ammonium and bicarbonate were absorbed simultaneously, the ammonium absorption occurred against a large transepithelial concentration gradient for NH3 and thus could not be explained by nonionic diffusion.! Most of the NH4 + absorption occurs by secondary active transport 23 ,24 and is mediated by substitution of NH4 + for K+ on the apical membrane Na +-K+ -2Cl- cotransport sys-

tem that mediates net NaCI reabsorption in thick ascending limbs (ie, by apical membrane Na +NH4 +-2CI- cotransport).1.23,25 The mechanism by which NH4 + exits the basolateral membrane has not been determined. In addition to the active, transcellular absorption, a minor fraction of NH4 + absorption occurs by paracellular diffusion, driven by the lumen-positive transepithelial voltage. 23,24 REGULATION

In the following sections, three factors that influence acid-base transport in the medullary thick ascending limb of the rat are discussed briefly to illustrate (1) that the thick ascending limb is a site of regulation of renal acid-base transport, and (2) that HC0 3- and NH4 + transport in this segment can vary independently. Potassium Concentration

Systemic potassium depletion generally is associated with increased urinary ammonium and net acid excretion, whereas potassium excess and hyperkalemia are associated with reduced ammonium and net acid excretion. 26 The relationship between potassium balance and ammonium excretion generally is attributed to effects of potassium on renal ammonium production. 26 ,27 An additional possibility, however, is that potassium could influence ammonium excretion by directly affecting renal tubule ammonium transport, independent of effects on ammonium production. 2! Figure 1 shows results of experiments designed to determine directly whether potassium influenced ammonium or bicarbonate transport in the medullary thick ascending limb of the rat in vitro. Increasing potassium concentration in the perfusion and bathing solutions did not significantly affect bicarbonate absorption but reduced ammonium absorption by 50 %. Further studies revealed that potassium inhibited specifically the active component of NH4 + absorption, most likely due to competition between NH4 + and K + for a common binding site on the apical Na +-K+ -2CIcotransport system.23 Based on these findings, it was proposed that systemic conditions that affect medullary potassium levels could influence urinary ammonium excretion by affecting NH4 + absorption in the medullary thick ascending limb and altering countercurrent multiplication of ammonium in the renal medulla. 21.23 For example, conditions that increase medullary potassium levels could reduce ammonium excretion by inhib-

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HC03- Absorption

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further studies, adding 8-Bromo-cyclic adenosine monophosphate (AMP) to the bath in the absence of added hormone also reduced He0 3 - absorption by 50%, 3 suggesting that the inhibition by vasopressin could have resulted from its action to stimulate adenylate cyclase and increase intracellular levels of cyclic AMP. This view was supported further by the observation that glucagon, another hormone that stimulates cyclic AMP production in the medullary thick ascending limb,29 also inhibited He0 3- absorption by approximately 50%.3 It appears therefore that peptide hormones and factors that affect intracellular levels of cyclic AMP may be important determinants of He0 3 - transport in the rat medullary thick ascending limb. The results obtained with vasopressin can be contrasted with those obtained in the potassium concentration experiments: vasopressin inhibited He0 3 - absorption with no detectable effect on NH4 + absorption, whereas an increase in K+ concentration inhibited NH4 + absorption with no effect on net He0 3 - absorption. These findings illustrate that, in the medullary thick ascending limb, NH4 + and He0 3 - absorption are subject to independent regulation.

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Perfusate and Bath [K+],mM Fig 1. Effects of potassium concentration on HC03 - and NH4 + absorption by the isolated, perfused rat medullary thick ascending limb. K+ concentrations (4 and 24 mmol/L) represent the physiological range of values expected for this segment in vivo. Values are means ± SE.• P < 0.001 (24 mmol/L K+ v 4 mmol/L K+); NS, no significant difference. (Data from references 21 and 23).

iting thick ascending limb ammonium absorption, reducing medullary ammonium recycling, and decreasing secretion of ammonium into the medullary collecting ducts. 21 Such effects could act in addition to effects of potassium on renal ammonium production to mediate changes in urinary ammonium excretion.

Vasopressin Peptide hormones such as vasopressin, glucagon, and parathyroid hormone regulate the transport of a number of important ions in thick ascending limbs. 28,29 These hormones also affect urinary net acid excretion and have been reported recently to influence reabsorption of bicarbonate along the superficial loop of Henle of the rat in vivo. 30 However, whether peptide hormones directly affect bicarbonate or ammonium transport in the thick ascending limb had not been determined. Figure 2 summarizes the effects of arginine vasopressin on acid-base transport by the rat medullary thick ascending limb in vitro. Vasopressin reduced bicarbonate absorption by 50 % but had no detectable effect on net ammonium absorption. In

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Fig 2. Effects of arginine vasopressin (AVP, 2 x 10-8 mol/L In the bath) on HC03 - and NH4 + absorption by the isolated, perfused rat medullary thick ascending limb. Values are means ± SE.• p < 0.005 (AVP v control [C)); NS, no significant difference. (Data from reference 3). The control ammonium absorption rate is less than that in Fig 1 due to use of a lower tubule perfusion rate (see reference 21).

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HC0 3-

Chronic Metabolic Acidosis

Chronic metabolic acidosis causes a marked stimulation of urinary net acid excretion, due largely to increased urinary excretion of ammonium. 19 Chronic acid loading is associated with an increase in the bicarbonate absorptive capacity of both the proximal tubule 15 and cortical collecting duct 6 .8 and greatly stimulates proximal tubule ammonium production and secretion. 18.19 The effects of chronic systemic metabolic acidosis on bicarbonate and ammonium transport in the medullary thick ascending limb are shown in Fig 3. In these studies, medullary thick ascending limbs from control and chronically acidotic rats were studied under identical conditions in vitro; thus, differences in transport rates represent adaptive changes in the intrinsic transport properties of the tubules. The results show that both bicarbonate and ammonium absorption were stimulated in medullary thick ascending limbs from acidotic rats. The adaptive changes observed in medullary thick ascending limb transport would aid in increasing net acid excretion during chronic metabolic acidosis. The increased intrinsic capacity for NH4 + absorption would contribute to the increase in loop ammonium absorption that occurs in acidoSiS 13 •19 and that may increase ammonium excretion by enhancing medullary ammonium recycling and increasing collecting duct ammonium secretion. 20 The increased capacity for He0 3 - absorption would help to maintain low luminal HC0 3 - concentrations along the loop of Henle and limit He0 3 - delivery to the distal tubule. This may be particularly important during chronic metabolic acidosis since increased NH4 + absorption along the medullary thick ascending limb would tend to alkalinize the loop fluid if the H+ secretory capacity was not simultaneously increased (direct NH4 + absorption removes a proton from the lumen and therefore would be equivalent to adding HC0 3 - to the tubule fluid). SUMMARY

The thick ascending limb of the rat influences urinary net acid excretion by reabsorbing both bicarbonate and ammonium. The HC0 3 - absorption is mediated by apical membrane Na +-H+ exchange; the NH4 + absorption is mediated predominantly by apical membrane Na +-NH/ -2Cl- co-

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Fig 3. Effects of chronic metabolic acidosis (CMA) on HC03 - and NH4 + absorption by the isolated, perfused rat medullary thick ascending limb. Acidotic rats drank 0.28 mollL NH4CI for 5 to 8 days; controls drank distilled H2 0. All rats received food ad libitum. Values are means ± SE .• p < 0.025 (CMA v control [Cl). (Data from references 4 and 5).

transport. In the medullary thick ascending limb in vitro, potassium concentration affects NH4 + but not HC0 3 - absorption, vasopressin affects He0 3 - but not NH4 + absorption, and chronic metabolic acidosis affects both He0 3 - and NH4 + absorption. Thus, in addition to segments of the proximal tubule and collecting duct, the thick ascending limb represents a site of regulation of renal acid-base transport. REFERENCES 1. Good DW, Knepper MA, Burg MB: Ammonia and bicarbonate transport by thick ascending limb of rat kidney. Am J Physiol 247:F35-F44, 1984 2. Good DW: Sodium-dependent bicarbonate absorption by cortical thick ascending limb of rat kidney. Am J Physiol 248:F821-F829, 1985 3. Good DW: Inhibition of bicarbonate absorption by peptide hormones in rat medullary thick ascending limb. Kidney Int 33:4(10, 1988 4. Good DW, Kurtz I: Effects of chronic metabolic acidosis and mineralocorticoid on H+ IHC0 3 - transport in rat medullary thick ascending limb. Kidney Int 35:454, 1989 (abstr) 5. Good DW, DuBose TD Jr: HC0 3 - and NH. + transport by rat medullary thick ascending limb: Effects of acid-base and Na + balance. Clin Res 37:581A, 1989 (abstr) 6. McKinney TD, Burg MB: Bicarbonate transport by rabbit cortical collecting tubules: Effect of acid and alkali loads in vivo on transport in vitro. J Clin Invest 60:766-768, 1977

266 7. Stone DK, Seldin DW, Kokko Jp, et al: Mineralocorticoid modulation of rabbit medullary collecting duct acidification. A sodium-independent effect. J Clin Invest 72:77-83, 1983 8. Atkins JL, Burg MB: Bicarbonate transport by isolated perfused rat collecting ducts. Am J Physiol 249:F485-F489, 1985 9. McKinney TD, Davidson KK: Bicarbonate transport in collecting tubules from outer stripe of outer medulla of rabbit kidneys. Am J Physiol 253:F816-F822, 1987 10. Wall SM, Sands JM, Knepper MA: Net acid secretion in isolated, perfused rat inner medullary collecting ducts. Kidney Int 35:465, 1989 (abstr) 11. Hays S, Kokko Jp, Jacobson HR: Hormonal regulation of proton secretion in rabbit medullary collecting duct. J Clin Invest 78:1279-1286, 1986 12. DuBose TD Jr, Lucci MS, Hogg RI, et al: Comparison of acidification parameters in superficial and deep nephrons of the rat. Am J Physiol 244:F497-F503, 1983 13. Buerkert J, Martin D, 'JHgg D: Segmental analysis of the renal tubule in buffer production and net acid formation. Am J Physiol 244:F442-F454, 1983 14. Krapf R: Basolateral membrane H/OH/HC0 3 transport in the rat cortical thick ascending limb. Evidence for an electrogenic NalHC0 3 cotransporter in parallel with a Na/H antiporter. J Clin Invest 82:234-241, 1988 15. Alpern RI, Warnock DG, Rector FC Jr: Renal acidification mechanisms, in Brenner BM, Rector FC Jr (eds): The Kidney. Philadelphia, PA, Saunders, 1986, pp 206-249 16. Guggino WB, Oberleithner H, Giebisch G: The amphibian diluting segment. Am J Physiol 254:F615-F627, 1988 17. Sajo 1M, Goldstein MB, Sonnenberg H, et al: Sites of ammonia addition to tubular fluid in rats with chronic metabolic acidosis. Kidney Int 20:353-358, 1981 18. Good DW, Knepper MA: Ammonia transport in the mammalian kidney. Am J Physiol 248:F459-F471, 1985

DAVIDWGOOD 19. Knepper MA, Packer R, Good DW: Ammonium transport in the kidney. Physiol Rev 69:179-249, 1989 20. Good DW, Caflisch CR, DuBose TD Jr: 'Iransepithelial ammonia concentration gradients in inner medulla of the rat. Am J Physiol 252:F491-F500, 1987 21. Good DW: Effects of potassium on ammonia transport by medullary thick ascending limb of the rat. J Clin Invest 80:1358-1365, 1987 22. DuBose TD Jr, Caflisch CR, Good DW: Effects of chronic hyperkalemia on renal ammonium transport. Kidney Int 35:452, 1989 (abstr) 23. Good DW: Active absorption of NH4 + by rat medullary thick ascending limb: Inhibition by potassium. Am J Physiol 255:F78-F87, 1988 24. Garvin JL, Burg MB, Knepper MA: Active NH4 + absorption by the thick ascending limb. Am J Physiol 255:F57F65, 1988 25. Kinne R, Kinne-Saffran E, Schutz H, et al: Ammonium transport in medullary thick ascending limb of rabbit kidney: Involvement of the Na+, K+, Cl- cotransporter. J Membr Bioi 94:279-284, 1986 26. Thnnen I,U.: Effect of potassium on renal acidification and acid-base homeostasis. Semin Nephrol 7:263-273, 1987 27. Thnnen RL: Relationship of renal ammonia production and potassium homeostasis. Kidney Int 11:453-465, 1977 28. Greger R: Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron. Physiol Rev 65:760-797, 1985 29. DeRouffignac C, Elalouf J, Roinel N: Physiological control of the urinary concentrating mechanism by peptide hormones. Kidney Int 31:611-620, 1987 30. Paillard M, Bichara M, Mercier 0, et al: Bicarbonate and chloride absorption in Henle's loop and the role of peptide hormones, in Davison AM (ed): Nephrology, vol 1. London, England, Bailliere Tindall, 1988, pp 275-280