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It Is Chloride Depletion , Not

Robert G. Luke and John H. Galla

Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio

ABSTRACT

Maintenance of generated by chloride depletion is often attributed aspiration, chloruretic diuretics, NaNO3 to . In balance and clearance studies in rats and humans, we infusion (an effect of un-reabsorbable showed that chloride repletion in the face of persisting alkali loading, volume anions), and prior hypercapnia (post- contraction, and potassium and sodium depletion completely corrects alkalosis hypercapnic CDA) were all used to gen- by a renal mechanism. Nephron segment studies strongly suggest the corrective erate CDA. These studies establish that 2 response is orchestrated in the collecting duct, which has several transporters Cl repletion by NaCl or KCl—but not integral to acid-base regulation, the most important of which is pendrin, a luminal replacement of Na+ and K+ losses without 2 2 Cl/HCO3 exchanger. Chloride depletion alkalosis should replace the notion of Cl —fully corrects CDA in the mainte- contraction alkalosis. nance phase. The issue of the specific role of ECF volume depletion was not re- J Am Soc Nephrol 23: 204–207, 2012. doi: 10.1681/ASN.2011070720 solved at this time. To separate chloride from volume re- pletion, we first studied rats with selective Chloridedepletionisthecommonestofthe identical to that in the plasma of the dogs CDA produced by peritoneal dialysis 3 three major causes of metabolic alkalosis; with stable alkalemia. These isometric in- against NaHCO3 and a normal serum po- theothersrelatetopotassiumdepletion/ fusions completely corrected alkalosis by tassium concentration. In rats given a 70 2 mineralocorticoid excess and to very low 24 hours without an increase in GFR, de- mEq/L Cl drink with either Na+ or cho- or absent glomerular filtration with base spite increasing potassium depletion; chlo- line, CDAwas completely corrected de- loading and are not further discussed in ride repletion was acknowledged. On the spite negative Na+ and K+ balances, detail. Metabolic alkalosis has generation, basis of the known characteristics of fluid decreased body weight, and obligatory maintenance, and recovery phases.1 This and electrolyte handling in the various sodium or choline bicarbonate loading.6 article focuses on the factors that affect nephronsegmentsatthattime,thevolume Acid-base status in choline-receiving con- the latter two phases. hypothesis in which the intranephronal re- trols was not altered. Rats treated in like In a seminal paper published in 1965, distribution of fluid reabsorption plays the manner with CDA maintained for 7–10 contraction alkalosis produced by etha- central role was expounded. ECF volume days responded in the same manner.7 crynic acid was described in humans that depletion accompanying alkalosis aug- The corrective response occurs by a renal gave rise to the hypothesis that extracel- ments fluidreabsorptionintheproximal mechanism.8 lular fluid (ECF) volume contraction pro- tubule where bicarbonate is preferentially To more rigorously exclude a role for duces alkalosis.2 The authors concluded reabsorbed compared with chloride; the volume expansion, CDA rats were infused that abrupt change in ECF volume was increased bicarbonate reabsorption in with 5% dextrose with either 6% albumin 2 the primary event, while acknowledging this segment thus maintains the alkalosis. or an isometric Cl solution. CDA that chloride depletion might influence During correction, volume expansion de- renal bicarbonate retention. creases proximal tubule fluid reabsorp- Published online ahead of print. Publication date In an effort to separate the correction tion, thereby delivering more bicarbonate available at www.jasn.org. of volume depletion from that of chloride to the distal nephron, which has a limited depletion, Cohen maintained alkalosis for capacity to reabsorb bicarbonate; bicarbon- Correspondence: Dr. Robert G. Luke, University of Cincinnati Medical Center, 231 Albert Sabin Way, 4 5 days in dogs treated with ethacrynic acid aturia ensues and the alkalosis corrects. PO Box 670556, Cincinnati OH 45267-0557. Email: and a NaCl-deficient diet, and then ex- Schwartz et al. studied the pathogen- [email protected] fl pandedECFvolumewitha uidcontaining esis of alkalosis produced by chloride de- Copyright © 2012 by the American Society of chloride and bicarbonate in concentrations pletion (CDA) in men and dogs.5 Gastric Nephrology

204 ISSN : 1046-6673/2302-204 J Am Soc Nephrol 23: 204–207, 2012 www.jasn.org SCIENCE IN RENAL MEDICINE persisted in rats infused with albumin de- secreting protons or bicarbonate and of CDA, pendrin is stimulated both by spite 15% ECF volume expansion, but was chloride bicarbonate exchange indepen- low chloride distal delivery22 and by corrected by the Cl--containing solu- dent of sodium was yet to come. metabolic alkalosis,20 including intra- tion despite persistent volume depletion The site at which this renal mainte- cellular alkalosis. In potassium deple- and decreased GFR.9 Delivery of chloride nance and correction of CDA occurs was tion metabolic alkalosis, a high serum to the collecting duct was not statistically addressed in micropuncture and micro- bicarbonate is maintained by intracellu- different between the groups but was ab- perfusion studies in our peritoneal di- lar in the renal tubular cells with solutely greater in rats that corrected. alysis rat model. First, GFR was inversely resulting increased bicarbonate reab- Urinary bicarbonate excretion increased correlated with the degree of CDA by sorption at several sites along the neph- as chloride was infused, whereas it de- tubuloglomerular feedback.12 Although ron. Pendrin is reduced in potassium creased further in the volume-expanded intact function of the proximal tubule depletion24; this suggests that the signal rats. The magnitude of bicarbonaturia and the loop of Henle are essential to the for increased activity for the luminal anion approximated the estimated amount of renal response, these segments have no exchanger is related to changes in intracel- chloride delivered to the collecting ducts. identifiable adaptive role in the corrective lular pH, rather than extracellular pH or Renal chloride conservation persisted response to chloride repletion in that glo- urine pH. Pendrin has been studied in until plasma chloride concentration re- merulotubular balance is maintained mice, rats, rabbits, and the gills of rays22 turned to normal.10 whether CDA is being corrected.8,9 Delivery (where it responds to changes in acid-base In normal human participants, we of chloride and bicarbonate out of the loop status on moving from sea to fresh water). turned to diuretic-induced CDA. Furose- of Henle was not different whether rats In renal pendrin null mice, serum + + 9,13 2 mide, Na ,K citrate supplementation, were maintaining or correcting CDA. HCO3 concentration is higher and 2 and dietary Cl restriction produced sta- In the distal convoluted tubule, alkalosis urine is less acidic than in controls.25 bly maintained CDA for 5 days that was can induce HCO3 secretion, which is in- Humans with Pendred syndrome (hypo- completely corrected thereafter by oral hibited by the removal of luminal chlo- thyroid goiter and deafness) seem to KCl, despite the presence of maintained ride; it is likely that such effects emanate have normal acid-base status. However, negative Na+ balance and plasma volume from the connecting tubule.14 two recent descriptions of a severe met- contraction (measured by the 131I albu- In perfused cortical collecting ducts, abolic alkalosis in a child, given thiazide 2 26 min space and plasma albumin concen- HCO3 is secreted in tubules from CDA for excess endolymph and severe alka- trations) and persistently lowered GFR rats and is dependent on luminal chlo- losis and potassium depletion in re- and estimated renal plasma flow.11 During ride, whereas it is reabsorbed in normal sponse to vomiting,27 suggest that an correction, net acid excretion decreased rats.15 The magnitude and direction of intact functioning of B-type intercalated 2 2 with HCO3 diuresis. In contrast, neutral HCO3 transport is dependent upon the cells are important for defense against sodium phosphate given in lieu of KCl degree of alkalosis and chloride repletion chloride depletion. was associated with increased serum in vivo at the time of tubule harvesting.16 The role of , if any, in 2 HCO3 concentration despite increased Thus, our studies in rats and humans regulating pendrin’s response to changes plasma volume. In control participants, suggest that the single and necessary in dietary chloride and to acid-base 2 furosemide administration without Cl correction effect for CDA is an increase changes is not clear. We showed that an- restriction did not cause CDA and serum in distal nephron chloride; the B-type giotensin II was not regulatory in main- electrolyte concentrations and net acid ex- intercalated cells along the cortical collect- tenance or correction of CDA.28 High cretion did not change with the same ingductwerepoisedtosecretebicarbonate aldosterone levels are also not necessary amount of KCl administration. Thus, in in exchange for administered chloride for maintenance of CDA29 and correction both rats and humans, chloride repletion (Figure 1). H+-ATPase activity was in- by chloride occurs despite rising levels.10 in the face of persistent volume depletion creased at the basolateral membrane of Aldosterone, within its physiologic range corrects CDA, whereas volume expansion B-type cells,17 but pendrin (the chloride of levels, is not important for direct without chloride does not. bicarbonate exchanger) was not yet iden- pendrin regulation.20,21 Adrenalecto- The contraction alkalosis viewpoint tified as the luminal anion exchanger. mized rats ingesting a low chloride diet was consistent with then-current views Abundant evidence now supports and drinking isotonic neutral sodium of the mechanism of renal bicarbonate pendrin as an important regulatory phosphate excrete extremely low concen- reabsorptionbysodium-protonexchange. transporter in the cortical collecting trations of chloride and maintain chloride Aldosterone was regarded as necessary to ducts, which responds briskly18 in vivo balance.30 Again, this suggests that chlo- increase thesodiumavidityofECFvolume and in vitro16 to alterations in chloride in- ride conservation in the distal nephron is contraction. Chloride was regarded as the take19 and to acid-base perturbations in- not aldosterone dependent. mendicant anion, allowing for sodium cluding metabolic alkalosis and acidosis20 Recently, a new mechanism of electro- reabsorption without obligating proton and respiratory acidosis21 (Table 1). The neutral sodium chloride absorption, secretion. Knowledge about specialized main stimuli seem to be distal chloride which is not inhibited by amiloride but cells in the distal nephron capable of delivery22 and intracellular pH.23 In is by thiazides, has been shown in the

J Am Soc Nephrol 23: 204–207, 2012 Chloride Depletion Alkalosis 205 SCIENCE IN RENAL MEDICINE www.jasn.org

and chloride is intensely conserved along the collecting ducts on a low chlo- ride diet.33 In summary, CDA can be corrected by selective chloride repletion despite main- tained or increasingly negative sodium or potassium balance, continued bicarbon- ate loading, and continuing high levels of angiotensin II or aldosterone, and is not corrected by sodium or potassium reple- tionwithoutchloriderepletion,bychloride repletion in the absence of renal function, by plasma volume expansion by as much as 15%, or by restoring baseline GFR without chloride repletion. In addition, we have demonstrated in vitro and in vivo correc- tion mechanisms by anion exchange along the collecting duct, which are consistent with current physiology. The more proxi- mal nephron segments do not contribute to the adaptive response for the correction of CDA by administered chloride. Chloride administration without vol- ume expansion is necessary and sufficient

2 to correct CDA. Volume deficits, whether Figure 1. In the CCD during reduced Cl delivery to that segment in CDA, pendrin activity is 2 - 2 associated with or al- increased but secretion of HCO3 is inhibited by insufficient Cl for anion exchange. HCO3 reabsorption may be partly maintained by Na+/H+ exchange by the electrical coupling be- kalosis, should be corrected. We suggest tween the principal cell and the H+-secreting A cell (although it is morphologically less active thatthetermchloridedepletionalkalosisis than normal17) and by the remainder of the collecting duct in the medulla. Coupled Na+/K+ clinically valuable because it indicates the 2 exchange causes kaliuresis. After Cl delivery to the cortical collecting ducts increases, correct pathophysiology and focuses on 2 2 32 HCO3 secretion occurs and medullary HCO3 reabsorption diminishes, allowing correc- the ion in the blood and urine that is tion of the hypochloremic alkalosis. In our rat studies, bicarbonaturia occurred within minutes fundamental to that mechanism. 2 2 of Cl administration intravenously and Cl did not increase in the urine until correction of 2 + 2 serum [Cl ] was nearly complete. (Both H and HCO3 are produced in the A- and B-type cells by carbonic anhydrase, and during acute acid-base changes, pendrin and H+ ATPase shuttle back and forth from cytosol to the luminal or basolateral membrane18). ACKNOWLEDGMENTS

Table 1. Responses of CCD luminal reduced chloride delivery to the cortical This study was supported by grants from the B cell pendrin to changes in dietary collecting ducts causes sodium conserva- National Institutes of Health. chloride and acid-base perturbations tion to be more dependent on ENaC and B Cell Pendrin may explain the high losses of urinary DISCLOSURES Condition Expression/ potassium in CDA. Activity Our conceptual model of mainte- None. Low chloride diet ↑ nance and correction of CDA is shown ↓ High chloride diet (Figure 1). In the rat model, bicarbonate REFERENCES Chloride depletion alkalosis ↑ secretion in exchange for administered Metabolic acidosis ↓ chloride in the cortical collecting duct ↓ 1. Seldin DW, Rector FC Jr: Symposium on acid- can account for correction of CDA. base . The generation and main- Potassium depletion ↓ Clearly there must be a coordinated re- tenance of metabolic alkalosis. Kidney Int 1: alkalosisa sponse in more distal sites to allow se- 306–321, 1972 a Associated with intracellular acidosis. creted bicarbonate to pass into the final 2. Cannon PJ, Heinemann HO, Albert MS, Laragh “ ” urine. We have shown in medullary col- JH, Winters RW: Contraction alkalosis after 31 diuresis of edematous patients with ethacrynic mouse cortical collecting duct. It is de- lecting duct segments that bicarbonate acid. Ann Intern Med 62: 979–990, 1965 pendent on pendrin and another sodium- reabsorption is decreased in tubules ob- 3. Cohen JJ: Correction of metabolic alkalosis dependent anion exchanger. Thus, tained from rats with correcting CDA32 by the kidney after isometric expansion of

206 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 204–207, 2012 www.jasn.org SCIENCE IN RENAL MEDICINE

extracellular fluid. JClinInvest47: 1181– bidirectional bicarbonate flux in vivo. JClin 24. Wagner CA, Finberg KE, Stehberger PA, Lifton 1192, 1968 Invest 85: 1793– 1798, 1990 RP, Giebisch GH, Aronson PS, Geibel JP: 4. Schwartz WB, Cohen JJ: The nature of the re- 15.GiffordJD,SharkinsK,WorkJ,LukeRG, Regulation of the expression of the Cl-/anion

nal response to chronic disorders of acid-base Galla JH: Total CO2 transport in rat cortical exchanger pendrin in mouse kidney by acid- equilibrium. Am J Med 64: 417–428, 1978 collecting duct in chloride-depletion al- base status. Kidney Int 62: 2109–2117, 2002 5. Schwartz WB, Kassirer JP Van Ypersele de kalosis. Am J Physiol 258: F848–F853, 25. Amlal H, Petrovic S, Xu J, Wang Z, Sun X, Strihou: Role of anions in metabolic alkalosis 1990 Barone S, Soleimani M: Deletion of the anion and potassium deficiency. NEnglJMed279: 16. Gifford JD, Ware MW, Luke RG, Galla JH: exchanger Slc26a4 (pendrin) decreases apical

630–639, 1968 HCO3- transport in rat CCD: Rapid adapta- Cl(-)/HCO3(-) exchanger activity and impairs 6. Galla JH, Bonduris DN, Luke RG: Correction tion by in vivo but not in vitro alkalosis. Am J bicarbonate secretion in kidney collecting of acute chloride-depletion alkalosis in the Physiol 264: F435–F440, 1993 duct. Am J Physiol Cell Physiol 299: C33–C41, rat without volume expansion. Am J Physiol 17. Verlander JW, Madsen KM, Galla JH, Luke 2010 244: F217–F221, 1983 RG, Tisher CC: Response of intercalated cells 26. Pela I, Bigozzi M, Bianchi B: Profound hypo- 7. Wall BM, Byrum GV, Galla JH, Luke RG: Im- to chloride depletion metabolic alkalosis. kalemia and hypochloremic metabolic alka- portance of chloride for the correction of Am J Physiol 262: F309–F319, 1992 losis during thiazide therapy in a child with chronic chloride depletion metabolic alkalo- 18. Purkerson JM, Tsuruoka S, Suter DZ, Nakamori Pendred syndrome. Clin Nephrol 69: 450– sis in the rat. Am J Physiol Renal Physiol 253: A, Schwartz GJ: Adaptation to metabolic aci- 453, 2008 F1031–F1039, 1987 dosis and its recovery are associated with 27. Kandasamy N, Fugazzola L, Evans M, 8. Craig DM, Galla JH, Bonduris DN, Luke RG: changes in anion exchanger distribution and Chatterjee K, Karet F: Life-threatening meta- Importance of the kidney in the correction of expression in the cortical collecting duct. Kid- bolic alkalosis in Pendred syndrome. Eur J chloride-depletion alkalosis in the rat. Am ney Int 78: 993–1005, 2010 Endocrinol 165: 167–170, 2011 JPhysiol250: F54–F57, 1986 19. Verlander JW, Kim YH, Shin W, Pham TD, 28. Walters EA, Rome L, Luke RG, Galla JH: Ab- 9. Galla JH, Bonduris DN, Luke RG: Effects of Hassell KA, Beierwaltes WH, Green ED, sence of a regulatory role of angiotensin II in chloride and extracellular fluid volume on bi- Everett L, Matthews SW, Wall SM: Dietary acute chloride-depletion alkalosis in the rat. carbonate reabsorption along the nephron in Cl(-) restriction upregulates pendrin expres- Am J Physiol 261: F741–F745, 1991 metabolic alkalosis in the rat. Reassessment sion within the apical plasma membrane of 29. Kassirer JP, Appleton FM, Chazan JA, Schwartz of the classical hypothesis of the pathogenesis type B intercalated cells. Am J Physiol Renal WB: Aldosterone in metabolic alkalosis. J Clin of metabolic alkalosis. J Clin Invest 80: Physiol 291: F833–F839, 2006 Invest 46: 1558–1571, 1967 41–50, 1987 20. Frische S, Kwon TH, Frøkiaer J, Madsen KM, 30. Luke RG: Effect of adrenalectomy on the re- 10. Galla JH, Bonduris DN, Dumbauld SL, Luke Nielsen S: Regulated expression of pendrin nal response to chloride depletion in the rat.

RG: Segmental chloride and fluid handling in rat kidney in response to chronic NH4Cl or JClinInvest54: 1329–1336, 1974

during correction of chloride-depletion al- NaHCO3 loading. Am J Physiol Renal Physiol 31. Leviel F, Hübner CA, Houillier P, Morla L, El kalosis without volume expansion in the rat. 284: F584–F593, 2003 Moghrabi S, Brideau G, Hassan H, Parker JClinInvest73: 96–106, 1984 21. de Seigneux S, Malte H, Dimke H, Frøkiaer J, MD, Kurth I, Kougioumtzes A, Sinning A, 11. Rosen RA, Julian BA, Dubovsky EV, Galla JH, Nielsen S, Frische S: Renal compensation to Pech V, Riemondy KA, Miller RL, Hummler E, Luke RG: On the mechanism by which chlo- chronic hypoxic hypercapnia: Downregulation Shull GE, Aronson PS, Doucet A, Wall SM, ride corrects metabolic alkalosis in man. of pendrin and adaptation of the proximal Chambrey R, Eladari D: The Na+-dependent Am J Med 84: 449–458, 1988 tubule. Am J Physiol Renal Physiol 292: chloride-bicarbonate exchanger SLC4A8 12. Galla JH, Bonduris DN, Sanders PW, Luke RG: F1256–F1266, 2007 mediates an electroneutral Na+ reabsorp- Volume-independent reductions in glomerular 22. Vallet M, Picard N, Loffing-Cueni D, Fysekidis tion process in the renal cortical collecting filtration rate in acute chloride-depletion alka- M, Bloch-Faure M, Deschênes G, Breton S, ducts of mice. JClinInvest120: 1627– losis in the rat. Evidence for mediation by tu- Meneton P, Loffing J, Aronson PS, Chambrey 1635, 2010 buloglomerular feedback. J Clin Invest 74: R, Eladari D: Pendrin regulation in mouse 32. Galla JH, Rome L, Luke RG: Bicarbonate 2002–2008, 1984 kidney primarily is chloride-dependent. JAm transport in collecting duct segments during 13. Galla JH, Bonduris DN, Luke RG: Superficial Soc Nephrol 17: 2153–2163, 2006 chloride-depletion alkalosis. Kidney Int 48: distal and deep nephrons in correction of 23. Adler L, Efrati E, Zelikovic I: Molecular 52–55, 1995 metabolic alkalosis. Am J Physiol 257: F107– mechanisms of epithelial cell-specificex- 33. Kirchner KA, Galla JH, Luke RG: Factors F113, 1989 pression and regulation of the human anion influencing chloride reabsorption in the col- 14. Levine DZ, Vandorpe D, Iacovitti M: Lumi- exchanger (pendrin) gene. Am J Physiol Cell lecting duct segment of the rat. Am J Physiol nal chloride modulates rat distal tubule Physiol 294: C1261–C1276, 2008 239: F552–F559, 1980

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