NEW ROLES FOR CHLORIDE IN RENAL PHYSIOLOGY AND PATHOPHYSIOLOGY* ROBERT G. LUKE,** and (by invitation) J. DANIEL GIFFORD and JOHN H. GALLA CINCINNATI
Until approximately thirty years ago, movement of chloride across epithelia was regarded as passive (secondary to active sodium transport) and usually paracellular. We now know that there are, in some cases via transmembrane chloride channels, transport processes in which movement of either chloride by itself or with other accompanying cations or anions play an essential role (1, 2). Transcellular movement occurs via active transmembrane and transepithelial chloride transport involving both reabsorption and secretion against electrochemical gradients. This is sometimes called "secondary active" transport since the electromotive force providing energy for uphill transport is usually ultimately provided by NaK ATPase or H ATPase. Examples include the NaK2Cl transporter in the luminal membrane of the thick ascending limb of the loop of Henle, the inhibition of which is responsible for the effect of loop diuretics (2); membrane chloride channels in bronchial epithelium the regulation of which is defective in cystic fibrosis (3); chloride-bicarbonate exchange via an anion exchanger in luminal and basolateral membranes of intercalated cells in the collecting duct (4); and KCI movement from cells into the extracellular fluid when they are subject to a hypotonic environment (1). Because of these specific transport effects, abnormalities of chloride transport are now recognized to play an important role in renal pathophysiology. About 25% of filtered sodium is reabsorbed as sodium chloride in the ascending limb of Henle's loop. Chloride delivery to and transepithelial reabsorption at or near the macula densa at the end of the thick ascending limb controls both renal renin release and tubulo-glomerular feedback. The latter mechanism is responsible for regulating single nephron GFR (glomerular filtration rate) in an attempt to ensure relative constancy of delivery of solute and fluid to the distal convoluted tubule and collecting duct-the sites of final adjustment of excretion of sodium, chloride, potassium, fluid and of acid base balance. Thus, high rates of * Department of Internal Medicine University of Cincinnati Medical Center Cincinnati, Ohio ** Address all correspondence to the author: Department of Internal Medicine, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0557.
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NaCl delivery to the macula densa in general are associated with reduction in GFR, and low rates are associated with an increase in GFR at the single nephron level via an effect on the afferent arteriolar tone (5). One plausible theory for the origin of Bartter's syndrome is a defect or abnormality in the NaK2CI luminal transporter in the thick ascending limb resulting in wasting of sodium, potassium and chloride and a marked stimulation of the renin-angiotensin-aldosterone axis (6). If this hypothesis is accurate, it is easy to see why the main differential diagnosis of Bartter's syndrome is abuse of diuretics acting on the thick ascending limb of Henle's loop. The anions accompanying sodium in states of deficit or excess of that cation also importantly influence the resulting effects. For example, in both animal models of essential hypertension and in hypertensive patients, sodium chloride loading, but not sodium bicarbonate loading, is associated with an increase in blood pressure (7). In an experimental anephric dog model, exchange of chloride for bicarbonate by hemoperfusion resulted in plasma volume contraction without (obviously) renal losses of fluid or sodium (8). Volume depletion in these circumstances may relate to shifts of fluid and electrolyte into cells as part of the buffering process or a direct effect of metabolic alkalosis on venous capacitance or arteriolar vasoconstriction. Because chloride and bicarbonate are the major extracellular fluid anions and because bicarbonate is a component of the major body buffering system, changes in the relative amounts of these anions are associated with acidosis and alkalosis, both as a primary acid base disturbance, and as a renal compensatory mechanism in response to primary respiratory acid-base disorders. Hyperchloremic metabolic acidosis is associated with bicarbonate wasting either in the feces or in urine. In acute respiratory acidosis, the kidney generates new bicarbonate for the extracellular fluid and during the compensatory phase has a negative chloride balance via chloruresis without a change in sodium balance (in the absence in any complicating problem such as congestive heart failure). Once the new steady state of hypochloremic hyperbicarbonatemia has developed chloride balance and net acid excretion resume appropriate balance. There is also a type of distal renal tubular acidosis which is believed to be caused by a chloride "shunt" in the collecting duct, resulting in hypertension due to sodium chloride retention. Hyperkalemia and acidosis relate to diminished sodium-potassium and sodium-proton exchange, since more of collecting duct sodium reabsorption is "shunted" to sodium chloride (9). In order to illustrate the importance of total body chloride in the pathophysiology of acid base disturbances, I would like to discuss the
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pathogenesis of chloride depletion metabolic alkalosis (CDA), a pathophysiological state in which my colleagues and I have been exploring the hypothesis that after the generation of metabolic alkalosis by, for example, vomiting or naso-gastric aspiration, the subsequent maintenance of metabolic alkalosis is explained by the state of chloride depletion per se by a renal effect to alter bicarbonate reabsorption. The classical view as to the mechanism underlying maintenance of metabolic alkalosis associated with chloride depletion, as outlined by Seldin (10) and Cohen (11) and others, placed the responsibility for enhanced reabsorption of bicarbonate in metabolic alkalosis on the associated volume depletion and accompanying renal tubular sodium avidity. Cogan has emphasized the role of GFR and has pointed out that in many circumstances GFR is reduced so that an increased tubular reabsorption of bicarbonate is not required for maintenance of alkalosis (12). As discussed below, we have addressed both the volume and the GFR hypothesis and in both animals and man have demonstrated that chloride depletion is the single and necessary effect maintaining CDA (13-20). Volume depletion and a depression of GFR are commonly associated, but not pathophysiologically responsible, factors. Indeed, there is evidence that chloride depletion, as discussed above, may itself be responsible for volume depletion, although in many circumstances responsible for CDA, such as vomiting, naso-gastric aspiration and the use of loop diuretics, volume depletion is present and related to external losses of sodium and fluid as well as of chloride. We have also shown that chloride depletion does produce a depression of GFR not dependent on volume contraction but related to tubulo-glomerular feedback (16). Our experimental approach was to use a rat model of CDA induced by peritoneal dialysis against sodium and potassium bicarbonate which resulted in selective depletion of chloride without sodium or potassium depletion. We maintained constant plasma volume by replacement with littermate blood of blood removed for measurements. Varying degrees of CDA are conveniently created by this technique by altering the length and number of dialyses; moreover 5% dextrose infusion without electrolytes can maintain a steady state of CDA for many hours. By a series of clearance and micropuncture studies, we have been able to show that chloride infusion without volume expansion, and indeed in the presence of increasing plasma volume contraction and without sodium administration, can correct CDA by a renal mechanism including increased bicarbonaturia. In a chronic model of CDA, produced by peritoneal dialysis and maintained by dietary chloride restriction for seven to ten days, similar findings were noted (19). As had been shown by Schwartz and coworkers many years before, CDA was corrected even as potassium balance became more negative (21).
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Although carefully controlled for changes in plasma volume, we noted a significant fall in GFR which was not explained by plasma volume contraction and the severity of which correlated with the degree of alkalosis. We showed that this was due to increasing tubulo-glomerular feedback due to increased delivery of solute, substantially sodium bicarbonate, to the macula densa. Since sodium bicarbonate is much less reabsorbable in the thick ascending limb of Henle's loop than is sodium chloride, the reduction of GFR by 30 or 40% considerably reduced losses of sodium bicarbonate in the urine in severe CDA until distal tubule reabsorptive mechanisms for bicarbonate reabsorption can become enhanced. The phase of biocarbonaturia, before maintenance alkalosis has developed, is termed disequilibrium alkalosis. In the chronic CDA model infusion of chloride without sodium led to correction of CDA despite a continuing decline in GFR. Since chloride replacement corrected the alkalosis despite volume contraction becoming more severe, the latter cannot be responsible for maintenance of CDA. We have thus demonstrated unequivocally that chloride depletion was the single and necessary effect for maintenance of CDA. We then proceeded to further explore the hypothesis in man. CDA was produced in normal volunteers in a clinical research center by a low salt diet supplemented by sodium and potassium citrate and by furosemide administration (22). As anticipated, CDA developed and was maintained by the low sodium chloride intake; there was also a steady state cumulative negative sodium balance. As had previously been shown by Schwartz and coworkers, oral KCI fully corrected the CDA despite the persistently negative cumulative sodium balance (23). What was new, however, is that we carefully followed plasma volume, GFR, effective renal plasma flow, serum protein concentration, hematocrit and cardiac output. Immediately after administration of KCI, bicarbonaturia occurred and fully accounted for the fall in plasma bicarbonate. Sodium balance, weight, plasma volume, GFR, renal plasma flow and serum protein concentration all remained significantly reduced as compared to baseline prior to induction of CDA. We also produced a very similar degree of sodium depletion, but without chloride depletion or CDA, by chloride replacement after furosemide and then gave the identical amount of KCI that corrected CDA in the previous experimental group. In this control group there were no changes in net acid excretion or in systemic acid base balance, demonstrating that the effect of KCI in the experimental group related to chloride repletion rather than to potassium loading. Overall these experiments in man nicely confirm that chloride repletion by a direct renal effect produced biocarbonaturia and fully corrected CDA despite a persistently negative sodium balance, reduction in GFR, weight and plasma volume. Thus despite persistent sodium avidity, the
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kidney is able to "exchange" chloride for bicarbonate in the renal tubule. We then proceeded to exlore further the renal mechanism by which chloride corrects CDA. We utilized the peritoneal dialysis rat model infused with isotonic dextrose with stable CDA and minimal bicarbonaturia. A control group continued with dextrose infusion and an experimental group was studied by micropuncture techniques during correction of CDA by infusion of chloride without sodium (as a chloride "cocktail" containing minimal amounts of calcium, magnesium, lithium and potassium which either did not induce detectable (lithium) or alter plasma levels of these cations). We measured chloride and bicarbonate delivery to the end of the accessible distal convoluted tubule of superficial nephrons (24). To measure delivery from the proximal segments of deep nephrons, we punctured the tip of the loop of Henle of deep nephrons, the only point at which they are accessible to micropuncture. Neither in deep or in superficial nephrons was there an increase in bicarbonate delivery to the point of puncture despite the considerable increase in bicarbonaturia in the cloride-infused, CDA-correcting group (Figure 1). These data are extremely suggestive ~~~~~c
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that the changes in bicarbonate excretion and chloride conservation responsible for correction of CDA were taking place in the collecting duct. The cortical collecting tubule of the rat collecting duct can only be studied by microdissection and in vitro microperfusion which Dr. Gifford then performed. It had been established in the preceding decade, mainly in the rabbit cortical collecting duct (CCD) but also in the rat (25), that this segment was capable of net bicarbonate secretion when the tubules were taken from animals previously loaded with oral sodium bicarbonate (but not necessarily with an actual sustained metabolic alkalosis). Morphological and functional studies had shown that there is cellular heterogeneity in the collecting duct (4). The CCD is made up of principal cells which absorb sodium and secrete potassium and are dependent on NaK ATPase, and intercalated cells of two types, A and B. The A-cell is a proton secreting cell with luminal H+ATPase and a basolateral chloride-bicarbonate exchanger; the B intercalated cell is responsible for bicarbonate secretion via luminal chloride bicarbonate exchange and a basolateral H+ATPase. The chloride bicarbonate exchanger is electro-neutral and, in the absence of luminal chloride, bicarbonate secretion ceases. We studied CCD's taken either from the peritoneal dialysis CDA model or from control rats subjected to peritoneal dialysis against Ringer's bicarbonate with maintained normal plasma electrolytes (26). In vitro, the control tubules absorbed bicarbonate and the experimental tubules secreted bicarbonate. Bicarbonate secretion persisted in vitro for approximately four hours despite the tubules being exposed in both lumen and bath to a symmetrical solution containing chloride at a concentration of 105 mM. Calculations, obtained by multiplying the net bicarbonate secretion rate in CDA rats in vitro by the average number of rat CCD's in kidneys, showed that the magnitude of this secretion was sufficient to account for the bicarbonaturia seen during correction in vivo. These estimates in no way prove that this is indeed the mechanism of correction, especially since we are comparing in vivo and in vitro experiments. Furthermore, there are segments distal to the CCD in the collecting duct which are capable of altering net bicarbonate reabsorption, although bicarbonate secretion itself is believed to be confined to the cortical collecting tubule. Recent morphological studies of our model by Dr. Tischer's group have provided additional evidence of inhibition of A-cell activity and activation of B-cell activity in the CCD of the CDA model during the early stages of correction by chloride (27). The basolateral infolding of the B-cells became more prominent and there was increased presence of H+ATPase in the basolateral membrane in the B-cells by the gold-conjugated double antigen technique. These morphological studies support the concept of a B-cell in CDA rats that it is "poised to secrete"
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bicarbonate. Urinary chloride remains virtually absent from the urine until correction of CDA is complete, suggesting uptake of chloride in the collecting duct and consistent with the concept of luminal chloridebicarbonate exchange. Some recent estimates of the Km for the luminal chloride exchanger in the rabbit (28) are consistent with the hypothesis that low chloride delivery rates in the collecting duct of chloride deplete animals would prevent bicarbonate secretion and maintain alkalosis until infusion of chloride and increased delivery of chloride to the cortical collecting tubule allowed bicarbonaturia to occur. In our micropuncture studies, delivery of chloride to the collecting duct did increase during correction of CDA but the magnitude of the increase was not statistically significant (24). Further work is required to confirm the hypothesis that B-cells are activated to secrete bicarbonate in states of CDA but inhibited from secreting until chloride delivery to the collecting duct is adequate. We were puzzled by the persistent secretion of bicarbonate for up to four hours in vitro and are currently studying this phenomenon. In all previous studies of bicarbonate secretion in the CCD bicarbonate in other laboratories, secretion has been sustained only for about 60 minutes. The state of chloride depletion alkalosis, as compared to bicarbonate loading, clearly "turns on" the bicarbonate secretory mechanism. Two hypotheses as to why bicarbonate secretion persisted in vitro were that cellular chloride depletion was not repleted in vitro despite the physiological concentrations of chloride in the basolateral or luminal bathing fluid, or that a necessary inhibitory hormonal or neurohumoral factor was present in vivo but absent in vitro. We excluded the first hypothesis by measuring intercalated cell chloride using the electron microprobe (29). There was no difference between intracellular chloride in control or CDA animals at the end of the experiment, suggesting full repletion of any cellular chloride depletion present at the start of the in vitro perfusion studies. We did determine in these experiments that intracellular chloride in intercalated cells was higher than that in principal cells by approximately 30 mEq/L, again confirming functional as well as structural differences between the principal and the intercalated cell. Our most current experiments examine the second hypothesis (30). Briefly, we partially corrected one group of CDA animals by chloride infusion in vivo and completely corrected CDA in a second group of animals by an increased amount of chloride infusion and perfused cortical collecting tubules taken from each of these two experimental groups (Figure 2). The partially corrected tubules demonstrated no net secretion or absorption of bicarbonate and complete correction was associated with the return of net absorption. Thus, in vivo, partial and complete correction was associated with appropriate changes in vitro in cortical collecting
91
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duct secretion, if indeed the mechanism of the chloride replacement is as we have hypothesized. We also attempted to induce secretion in vitro by perfusion of tubules in a medium with chloride and bicarbonate concentrations and pH consistent with those of alkalotic blood in vivo, but were unsuccessful in inducing secretion in vitro in these circumstances. Thus, we believe that in vivo events turn off bicarbonate secretion in the CCD during correction of alkalosis but that this mechanism is absent in vitro. It is possibly relevant that ,B agonists stimulate in vitro secretion in the rabbit (4) and that vasointestinal peptide (VIP) stimulates bicarbonate secretion in the turtle bladder which has intercalated cells similar to those in the CCD (31). In summary, we believe that we have demonstrated that chloride is the single and necessary mechanism for the maintenance of CDA and that chloride repletion results in bicarbonate secretion via luminal chloride bicarbonate exchange in the B intercalated cell in the cortical collecting duct. The details on the control mechanisms for chloride bicarbonate exchange remain to be further elucidated. Further support for the possibly important role of chloride bicarbonate exchange in the collecting duct in maintaining systemic acid base balance
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is provided by evidence in man (32) and the rat (personal communication, Wall, Galla and Luke) that sodium bicarbonate loading produces metabolic alkalosis in circumstances of dietary chloride restriction but not when chloride intake is normal. It has also been noted that neonatal rabbits lack fully developed B-cells and have a mild metabolic alkalosis until these B intercalated cells mature (33). The specific effects of alterations in total body chloride and of alterations in chloride delivery rates and transport processes in various segments of the nephron are numerous and still being discovered. Chloridebicarbonate exchange in the collecting duct provides the kidney a mechanism for acid-base adjustments independent of sodium transport and excretion. REFERENCES 1. Aronson PS. Distribution of Sodium Chloride Across Cell Membranes. In Seldin DW, Giebisch G, eds. The Regulation of Sodium and Chloride Balance. New York: Raven; 1990: 3. 2. Koeppen BM. Mechanisms of Segmental Sodium and Chloride Reabsorption. In Seldin DW, Giebisch G, eds. The Regulation of Sodium and Chloride Balance. New York: Raven; 1990: 59. 3. Kirk KL. Defective Regulation of Epithelial Cl- Permeability and Protein Secretion in Cystic Fibrosis: The Putative Basic Defect. Am J Kidney Dis 1989; XIV: 333. 4. Koeppen BM, Giebisch G. Segmental Hydrogen Ion Transport. In Seldin DW, Giebisch G, eds. The Regulation of Acid-Base Balance. New York: Raven; 1989: 139. 5. Wright FS, Briggs JP. Feedback regulation of glomerular filtration rate. Am J Physiol 1977; 233: Fl. 6. Galla JH, Luke RG. Pathophysiology of Metabolic Alkalosis. Hosp Prac 1987: 123. 7. Kurtz TW, Al-Bander HA, Morris RC. "Salt-sensitive" essential hypertension in men. Is the sodium ion alone important? N Engl J Med 1987; 317: 1043. 8. Garella S, Northrup TE, Cohen JJ. Chloride-depletion metabolic alkalosis (CDMA) induces ECF volume (ECFV) contraction via internal fluid shifts in nephrectomized dogs. Kidney Int 1988; 33: 400. 9. Schambelan M, Sebastian A, Rector FC. Mineralocorticoid-resistant renal hyperkalemia without salt wasting (type II pseudohypoaldosteronism): Role of increased renal chloride reabsorption. Kidney Int New York: Raven; 1981; 716. 10. Emmett M, Seldin DW. Clinical syndromes of metabolic acidosis and metabolic alkalosis. In Seldin DW, Giebisch G, eds. The Kidney, Physiology and Pathophysiology. New York: Raven; 1985: 1611. 11. Cohen JJ. Correction of metabolic alkalosis by the kidney after isometric expansion of extracellular fluid. J Clin Invest 1968; 47: 1181. 12. Cogan MG, Liu FY. Metabolic alkalosis in the rat. Evidence that reduced glomerular filtration rate rather than enhanced tubular bicarbonate reabsorption is responsible for maintaining the alkalotic state. J Clin Invest 1983; 71: 1141. 13. Luke RG, Galla JH. Chloride-depletion alkalosis with a normal extracellular fluid volume. Am J Physiol 1983; 245: F419. 14. Galla JH, Bonduris DN, Luke RG. Correction of acute chloride-depletion alkalosis in the rat without volume expansion. Am J Physiol 1983; 244: F217.
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15. Galla JH, Bonduris DN, Dumbauld SL, et al. Segmental chloride and fluid handling during correction of chloride-depletion alkalosis without volume expansion in the rat. J Clin Invest 1984; 73: 96. 16. Galla JH, Bonduris DN, Sanders PW, et al. Volume-independent reductions in glomerular filtration rate in acute chloride-depletion alkalosis in the rat. J Clin Invest 1984; 74: 2002. 17. Galla JH, Bonduris DN, Luke RG. Effects of chloride and extracellular fluid volume on bicarbonate reabsorption along the nephron in metabolic alkalosis in the rat. J Clin Invest 1987; 80: 41. 18. Galla JH, Luke RG. Chloride transport and disorders of acid-base balance. Ann Rev Physiol 1988; 50: 141. 19. Wall BM, Byrum GV, Galla JH, et al. Importance of chloride for the correction of chronic chloride depletion metabolic alkalosis in the rat. Am J Physiol 1987; 253: F1031. 20. Craig DM, Galla JH, Bonduris DN, et al. Importance of the kidney in the correction of chloride-depletion alkalosis in the rat. Am J Physiol 1986; 250: F54. 21. Kassirer JP, Schwartz WB. Correction of metabolic alkalosis in man without repair of potassium deficiency. A re-evaluation of the role of potassium. Am J Med 1966; 40: 19. 22. Rosen RA, Julian BA, Dubovsky EA, et al. On the mechanism by which chloride corrects metabolic alkalosis in man. Am J Med 1988; 84: 449. 23. Kassirer JP, Schwartz WB. The response of normal man to selective depletion of hydrochloric acid. Factors in the genesis of persistent gastric alkalosis. Am J Med 1966; 40: 10. 24. Galla JH, Bonduris DN, Luke RG. Superficial distal and deep nephrons in the correction of metabolic alkalosis. Am J Physiol 1989 257: F107. 25. Atkins JL, Burg MB. Bicarbonate transport by isolated perfused rat collecting ducts. Am J Physiol 1985; 249: F485. 26. Gifford JD, Sharkins K, Work J, et al. Total CO2 transport in rat cortical collecting duct in chloride-depletion alkalosis. Am J Physiol 1990; 258: F848. 27. Verlander JW, Madsen KM, Galla JH, et al. Ultrastructural Localization of H+ATPase in Rat Cortical Collecting Duct (CCD) During Chloride Depletion Metabolic Alkalosis (CDA). JASN 1990; 1: 659. (Abstract) 28. Furuya M, Breyer M, Jacobson H. Functional Characterization of a and : Intercalated Cell Types in the Rabbit Cortical Collecting Duct. JASN 1990; 1: 649. (Abstract) 29. Gifford JD, Galla JH, Luke RG, et al. Ion concentrations in the rat cortical collecting duct: differences between cell types and effect of alkalosis. Am J Physiol 1990; 259: F778. 30. Gifford JD, Sharkins K, Luke RG, et al. Metabolic Alkalosis: in vivo Cl- Repletion Correlates with Cortical Collecting Duct Total CO2 Transport in vitro. JASN 1990; 1: 649. (Abstract) 31. Durham JH, Matons C, Brodsky WA. Vasoactive intestinal peptide stimulates alkali excretion in turtle urinary bladder. Am J Physiol 1987; (Cell Physiol 21) 252: C428. 32. Cogan MG, Carneiro J, Tatsuno J. Normal Diet NaCl Variation Can Affect the Renal Set-Point for Plasma pH-(HCO3) Maintenance. JASN 1990; 1: 193. 33. Satlin LM, Schwartz GJ. Postnatal Maturation of Intercalcated Cells (ICs) in the Rabbit cortical Collecting Duct (CCD). JASN 1990; 1-658. (Abstract)
DISCUSSION Christy (New York): May I exercise my privilege as Chairman to ask you one question if you can answer it simply. I'm afraid maybe you can't, but in the syndrome of so-called
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inappropriate ADH secretion, can you give us some quantitative idea of the waste of chloride vs sodium? Luke: In SIADH sodium and chloride are reduced proportionately mainly because of water retention rather than urinary losses of sodium and chloride. The great interest in the syndrome is why the serum bicarbonate does not fall. Schwartz did some beautiful studies where he showed that aldo levels, in fact, rose ultimately with low sodium and it may be there is a stimulating distal mechanism, that maintains what is a relative alkalosis in SIADH. So I am not answering your question, I am answering a related one that the interesting thing in this syndrome is not the hypochloremia which is proportionate to the sodium, but why does the bicarbonate stay normal. Christy (New York): How about potassium? Luke: The potassium tends to fall a little bit, but remains and falls in proportion, in my experience, to the sodium and chloride. Ferris (Minneapolis): Robin, all us younger Nephrologists accept the fact that chloride is actively handled by the kidneys. It's intriguing though, that with cystic fibrosis, where there is a defect in chloride absorption and so many aberrations in terms of secretions from the lung, there's never been any evidence that their renal physiological mechanism isn't intact. They dilute normally, and their renin secretion seems normal. Luke: The main new idea in cystic fibrosis is that regulation of the epithelial chloride channel in the chloride secreting bronchial apical cell is abnormal. (Kirk KL: Defective regulation of epithelial CL permeability and protein secretion in cystic fibrosis: the putative basic defect. Am J Kidney Dis 1989; 14:333) This causes the viscous bronchial secretions and subsequent respiratory problems and suggests that we may be able to restore normal regulation by drug therapy rather than perhaps having to use gene therapy. Rochester (Charlottesville): As a Pulmonologist, I recall a number of papers from the late 1950's that emphasized that in the recovery from hypercapnia respiratory failure, it was necessary to provide chloride and potassium and the questions that I wanted to ask you first were: in your rat model, does Diamox work if you don't replete chloride? And the other question is when you make your rats alkalotic, do they retain C02? Luke: They retain CO2 and I think that in animals and man CO2 retention in proportion to metabolic alkalosis is unequivocal. I think there is a 0.6 to 0.7 delta increase in CO2 for each milliequivalent increase in serum bicarbonate. I didn't use Diamox in the rats. In my mind, I think you simply compound the disturbance and make a metabolic acidosis as well as a metabolic alkalosis. You're talking about a cor pulmonale patient who is edematous and you can't give him sodium chloride and whose potassium is often high; I think you can give them Diomox and KCL, the two have to be combined. Dunn (Cleveland): That was an elegant summary. There are many parallels between what you described in patients with Bartter's syndrome and yet the putative defective site in Bartter's syndrome is quite different than the site that you focused on for the chloride biocarbonate exchange in the more distal nephron. Would you comment on that? Luke: Bartter's syndrome is not a pure chloride depletion alkalosis but rather is a mixed potassium depletion-aldosterone excess and chloride depletion alkalosis. One might call the syndrome "congenital lasixemia": the NAK2Cl transporter mechanism is likely abnormal in the thick ascending limb. Thus my concept about the Bartter's syndrome is that the collecting duct seems to be doing pretty well and that the defect is in the thick ascending limb which results in increased delivery of sodium potassium and chloride that overwhelms the collecting duct. J. Stein believes, in contrast, that the syndrome may be due to primary K wasting, and indeed there may be several mechanisms resulting in the syndrome. Chloride depletion alkalosis and Bartter's syndrome are clearly different, and Bartter's syndrome is an alkalosis of mixed etiology. Earle (Chicago): This is an old geezer question. Almost 40 years ago, Farber, Pellegrino,
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Alexander and I studied renal function and electrolyte metabolism in twelve episodes of acute glomerulonephritis in adults. We observed a moderate, but significant hyperchloremia. At the time, the patients were edematous, had slightly low serum sodium and were excreting excess amounts of ammonium chloride. This hyperchloremia persisted for weeks or months after everything else except abnormal urine had returned to normal. Could I ask you to explain this now? We had no explanation. Luke: This was hyperchloremic alkalosis. You know classically in chronic renal failure, the attitude has been that you get an anion gap acidosis early on. It's now quite clear that with creatinine of 2, 3, 4 and GFR's perhaps 20-50-60, the first change you get with progressive nephron loss is hyperchloremic, non-anion gap acidosis. The mechanism remains unknown. Now, did your patients have renal insufficiency? Earle (Chicago): Mild to moderate, and transient. Luke: I think that's the answer then. What's in some textbooks at the moment is that chronic renal failure is associated with anion gap acidosis like so many other things in the textbooks that someone else said. This is wrong, and if you really look at it carefully a nonanion gap acidosis is common. Indeed some people, including Jordan Cohen, have postulated that chloride transport is abnormal in some way in early chronic renal failure before bicarbonate transport is abnormal. It remains unknown why non-anion gap hyperchloremic acidosis is the first acidosis acquired by most patients with chronic renal failure. Only later do they get an anion gap acidosis. Earle (Chicago): These were acute. Luke: But you were following them over some weeks weren't you? Earle (Chicago): Yes, that's correct.
Until approximately thirty years ago, movement of chloride across epithelia was regarded as passive (secondary to active sodium transport) and usually paracellular. We now know that there are, in some cases via transmembrane chloride channels, transport processes in which movement of either chloride by itself or with other accompanying cations or anions play an essential role (1, 2). Transcellular movement occurs via active transmembrane and transepithelial chloride transport involving both reabsorption and secretion against electrochemical gradients. This is sometimes called "secondary active" transport since the electromotive force providing energy for uphill transport is usually ultimately provided by NaK ATPase or H ATPase. Examples include the NaK2Cl transporter in the luminal membrane of the thick ascending limb of the loop of Henle, the inhibition of which is responsible for the effect of loop diuretics (2); membrane chloride channels in bronchial epithelium the regulation of which is defective in cystic fibrosis (3); chloride-bicarbonate exchange via an anion exchanger in luminal and basolateral membranes of intercalated cells in the collecting duct (4); and KCI movement from cells into the extracellular fluid when they are subject to a hypotonic environment (1). Because of these specific transport effects, abnormalities of chloride transport are now recognized to play an important role in renal pathophysiology. About 25% of filtered sodium is reabsorbed as sodium chloride in the ascending limb of Henle's loop. Chloride delivery to and transepithelial reabsorption at or near the macula densa at the end of the thick ascending limb controls both renal renin release and tubulo-glomerular feedback. The latter mechanism is responsible for regulating single nephron GFR (glomerular filtration rate) in an attempt to ensure relative constancy of delivery of solute and fluid to the distal convoluted tubule and collecting duct-the sites of final adjustment of excretion of sodium, chloride, potassium, fluid and of acid base balance. Thus, high rates of * Department of Internal Medicine University of Cincinnati Medical Center Cincinnati, Ohio ** Address all correspondence to the author: Department of Internal Medicine, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0557.
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NaCl delivery to the macula densa in general are associated with reduction in GFR, and low rates are associated with an increase in GFR at the single nephron level via an effect on the afferent arteriolar tone (5). One plausible theory for the origin of Bartter's syndrome is a defect or abnormality in the NaK2CI luminal transporter in the thick ascending limb resulting in wasting of sodium, potassium and chloride and a marked stimulation of the renin-angiotensin-aldosterone axis (6). If this hypothesis is accurate, it is easy to see why the main differential diagnosis of Bartter's syndrome is abuse of diuretics acting on the thick ascending limb of Henle's loop. The anions accompanying sodium in states of deficit or excess of that cation also importantly influence the resulting effects. For example, in both animal models of essential hypertension and in hypertensive patients, sodium chloride loading, but not sodium bicarbonate loading, is associated with an increase in blood pressure (7). In an experimental anephric dog model, exchange of chloride for bicarbonate by hemoperfusion resulted in plasma volume contraction without (obviously) renal losses of fluid or sodium (8). Volume depletion in these circumstances may relate to shifts of fluid and electrolyte into cells as part of the buffering process or a direct effect of metabolic alkalosis on venous capacitance or arteriolar vasoconstriction. Because chloride and bicarbonate are the major extracellular fluid anions and because bicarbonate is a component of the major body buffering system, changes in the relative amounts of these anions are associated with acidosis and alkalosis, both as a primary acid base disturbance, and as a renal compensatory mechanism in response to primary respiratory acid-base disorders. Hyperchloremic metabolic acidosis is associated with bicarbonate wasting either in the feces or in urine. In acute respiratory acidosis, the kidney generates new bicarbonate for the extracellular fluid and during the compensatory phase has a negative chloride balance via chloruresis without a change in sodium balance (in the absence in any complicating problem such as congestive heart failure). Once the new steady state of hypochloremic hyperbicarbonatemia has developed chloride balance and net acid excretion resume appropriate balance. There is also a type of distal renal tubular acidosis which is believed to be caused by a chloride "shunt" in the collecting duct, resulting in hypertension due to sodium chloride retention. Hyperkalemia and acidosis relate to diminished sodium-potassium and sodium-proton exchange, since more of collecting duct sodium reabsorption is "shunted" to sodium chloride (9). In order to illustrate the importance of total body chloride in the pathophysiology of acid base disturbances, I would like to discuss the
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pathogenesis of chloride depletion metabolic alkalosis (CDA), a pathophysiological state in which my colleagues and I have been exploring the hypothesis that after the generation of metabolic alkalosis by, for example, vomiting or naso-gastric aspiration, the subsequent maintenance of metabolic alkalosis is explained by the state of chloride depletion per se by a renal effect to alter bicarbonate reabsorption. The classical view as to the mechanism underlying maintenance of metabolic alkalosis associated with chloride depletion, as outlined by Seldin (10) and Cohen (11) and others, placed the responsibility for enhanced reabsorption of bicarbonate in metabolic alkalosis on the associated volume depletion and accompanying renal tubular sodium avidity. Cogan has emphasized the role of GFR and has pointed out that in many circumstances GFR is reduced so that an increased tubular reabsorption of bicarbonate is not required for maintenance of alkalosis (12). As discussed below, we have addressed both the volume and the GFR hypothesis and in both animals and man have demonstrated that chloride depletion is the single and necessary effect maintaining CDA (13-20). Volume depletion and a depression of GFR are commonly associated, but not pathophysiologically responsible, factors. Indeed, there is evidence that chloride depletion, as discussed above, may itself be responsible for volume depletion, although in many circumstances responsible for CDA, such as vomiting, naso-gastric aspiration and the use of loop diuretics, volume depletion is present and related to external losses of sodium and fluid as well as of chloride. We have also shown that chloride depletion does produce a depression of GFR not dependent on volume contraction but related to tubulo-glomerular feedback (16). Our experimental approach was to use a rat model of CDA induced by peritoneal dialysis against sodium and potassium bicarbonate which resulted in selective depletion of chloride without sodium or potassium depletion. We maintained constant plasma volume by replacement with littermate blood of blood removed for measurements. Varying degrees of CDA are conveniently created by this technique by altering the length and number of dialyses; moreover 5% dextrose infusion without electrolytes can maintain a steady state of CDA for many hours. By a series of clearance and micropuncture studies, we have been able to show that chloride infusion without volume expansion, and indeed in the presence of increasing plasma volume contraction and without sodium administration, can correct CDA by a renal mechanism including increased bicarbonaturia. In a chronic model of CDA, produced by peritoneal dialysis and maintained by dietary chloride restriction for seven to ten days, similar findings were noted (19). As had been shown by Schwartz and coworkers many years before, CDA was corrected even as potassium balance became more negative (21).
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Although carefully controlled for changes in plasma volume, we noted a significant fall in GFR which was not explained by plasma volume contraction and the severity of which correlated with the degree of alkalosis. We showed that this was due to increasing tubulo-glomerular feedback due to increased delivery of solute, substantially sodium bicarbonate, to the macula densa. Since sodium bicarbonate is much less reabsorbable in the thick ascending limb of Henle's loop than is sodium chloride, the reduction of GFR by 30 or 40% considerably reduced losses of sodium bicarbonate in the urine in severe CDA until distal tubule reabsorptive mechanisms for bicarbonate reabsorption can become enhanced. The phase of biocarbonaturia, before maintenance alkalosis has developed, is termed disequilibrium alkalosis. In the chronic CDA model infusion of chloride without sodium led to correction of CDA despite a continuing decline in GFR. Since chloride replacement corrected the alkalosis despite volume contraction becoming more severe, the latter cannot be responsible for maintenance of CDA. We have thus demonstrated unequivocally that chloride depletion was the single and necessary effect for maintenance of CDA. We then proceeded to further explore the hypothesis in man. CDA was produced in normal volunteers in a clinical research center by a low salt diet supplemented by sodium and potassium citrate and by furosemide administration (22). As anticipated, CDA developed and was maintained by the low sodium chloride intake; there was also a steady state cumulative negative sodium balance. As had previously been shown by Schwartz and coworkers, oral KCI fully corrected the CDA despite the persistently negative cumulative sodium balance (23). What was new, however, is that we carefully followed plasma volume, GFR, effective renal plasma flow, serum protein concentration, hematocrit and cardiac output. Immediately after administration of KCI, bicarbonaturia occurred and fully accounted for the fall in plasma bicarbonate. Sodium balance, weight, plasma volume, GFR, renal plasma flow and serum protein concentration all remained significantly reduced as compared to baseline prior to induction of CDA. We also produced a very similar degree of sodium depletion, but without chloride depletion or CDA, by chloride replacement after furosemide and then gave the identical amount of KCI that corrected CDA in the previous experimental group. In this control group there were no changes in net acid excretion or in systemic acid base balance, demonstrating that the effect of KCI in the experimental group related to chloride repletion rather than to potassium loading. Overall these experiments in man nicely confirm that chloride repletion by a direct renal effect produced biocarbonaturia and fully corrected CDA despite a persistently negative sodium balance, reduction in GFR, weight and plasma volume. Thus despite persistent sodium avidity, the
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ROBERT G. LUKE
kidney is able to "exchange" chloride for bicarbonate in the renal tubule. We then proceeded to exlore further the renal mechanism by which chloride corrects CDA. We utilized the peritoneal dialysis rat model infused with isotonic dextrose with stable CDA and minimal bicarbonaturia. A control group continued with dextrose infusion and an experimental group was studied by micropuncture techniques during correction of CDA by infusion of chloride without sodium (as a chloride "cocktail" containing minimal amounts of calcium, magnesium, lithium and potassium which either did not induce detectable (lithium) or alter plasma levels of these cations). We measured chloride and bicarbonate delivery to the end of the accessible distal convoluted tubule of superficial nephrons (24). To measure delivery from the proximal segments of deep nephrons, we punctured the tip of the loop of Henle of deep nephrons, the only point at which they are accessible to micropuncture. Neither in deep or in superficial nephrons was there an increase in bicarbonate delivery to the point of puncture despite the considerable increase in bicarbonaturia in the cloride-infused, CDA-correcting group (Figure 1). These data are extremely suggestive ~~~~~c
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NEW ROLES FOR CHLORIDE
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that the changes in bicarbonate excretion and chloride conservation responsible for correction of CDA were taking place in the collecting duct. The cortical collecting tubule of the rat collecting duct can only be studied by microdissection and in vitro microperfusion which Dr. Gifford then performed. It had been established in the preceding decade, mainly in the rabbit cortical collecting duct (CCD) but also in the rat (25), that this segment was capable of net bicarbonate secretion when the tubules were taken from animals previously loaded with oral sodium bicarbonate (but not necessarily with an actual sustained metabolic alkalosis). Morphological and functional studies had shown that there is cellular heterogeneity in the collecting duct (4). The CCD is made up of principal cells which absorb sodium and secrete potassium and are dependent on NaK ATPase, and intercalated cells of two types, A and B. The A-cell is a proton secreting cell with luminal H+ATPase and a basolateral chloride-bicarbonate exchanger; the B intercalated cell is responsible for bicarbonate secretion via luminal chloride bicarbonate exchange and a basolateral H+ATPase. The chloride bicarbonate exchanger is electro-neutral and, in the absence of luminal chloride, bicarbonate secretion ceases. We studied CCD's taken either from the peritoneal dialysis CDA model or from control rats subjected to peritoneal dialysis against Ringer's bicarbonate with maintained normal plasma electrolytes (26). In vitro, the control tubules absorbed bicarbonate and the experimental tubules secreted bicarbonate. Bicarbonate secretion persisted in vitro for approximately four hours despite the tubules being exposed in both lumen and bath to a symmetrical solution containing chloride at a concentration of 105 mM. Calculations, obtained by multiplying the net bicarbonate secretion rate in CDA rats in vitro by the average number of rat CCD's in kidneys, showed that the magnitude of this secretion was sufficient to account for the bicarbonaturia seen during correction in vivo. These estimates in no way prove that this is indeed the mechanism of correction, especially since we are comparing in vivo and in vitro experiments. Furthermore, there are segments distal to the CCD in the collecting duct which are capable of altering net bicarbonate reabsorption, although bicarbonate secretion itself is believed to be confined to the cortical collecting tubule. Recent morphological studies of our model by Dr. Tischer's group have provided additional evidence of inhibition of A-cell activity and activation of B-cell activity in the CCD of the CDA model during the early stages of correction by chloride (27). The basolateral infolding of the B-cells became more prominent and there was increased presence of H+ATPase in the basolateral membrane in the B-cells by the gold-conjugated double antigen technique. These morphological studies support the concept of a B-cell in CDA rats that it is "poised to secrete"
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ROBERT G. LUKE
bicarbonate. Urinary chloride remains virtually absent from the urine until correction of CDA is complete, suggesting uptake of chloride in the collecting duct and consistent with the concept of luminal chloridebicarbonate exchange. Some recent estimates of the Km for the luminal chloride exchanger in the rabbit (28) are consistent with the hypothesis that low chloride delivery rates in the collecting duct of chloride deplete animals would prevent bicarbonate secretion and maintain alkalosis until infusion of chloride and increased delivery of chloride to the cortical collecting tubule allowed bicarbonaturia to occur. In our micropuncture studies, delivery of chloride to the collecting duct did increase during correction of CDA but the magnitude of the increase was not statistically significant (24). Further work is required to confirm the hypothesis that B-cells are activated to secrete bicarbonate in states of CDA but inhibited from secreting until chloride delivery to the collecting duct is adequate. We were puzzled by the persistent secretion of bicarbonate for up to four hours in vitro and are currently studying this phenomenon. In all previous studies of bicarbonate secretion in the CCD bicarbonate in other laboratories, secretion has been sustained only for about 60 minutes. The state of chloride depletion alkalosis, as compared to bicarbonate loading, clearly "turns on" the bicarbonate secretory mechanism. Two hypotheses as to why bicarbonate secretion persisted in vitro were that cellular chloride depletion was not repleted in vitro despite the physiological concentrations of chloride in the basolateral or luminal bathing fluid, or that a necessary inhibitory hormonal or neurohumoral factor was present in vivo but absent in vitro. We excluded the first hypothesis by measuring intercalated cell chloride using the electron microprobe (29). There was no difference between intracellular chloride in control or CDA animals at the end of the experiment, suggesting full repletion of any cellular chloride depletion present at the start of the in vitro perfusion studies. We did determine in these experiments that intracellular chloride in intercalated cells was higher than that in principal cells by approximately 30 mEq/L, again confirming functional as well as structural differences between the principal and the intercalated cell. Our most current experiments examine the second hypothesis (30). Briefly, we partially corrected one group of CDA animals by chloride infusion in vivo and completely corrected CDA in a second group of animals by an increased amount of chloride infusion and perfused cortical collecting tubules taken from each of these two experimental groups (Figure 2). The partially corrected tubules demonstrated no net secretion or absorption of bicarbonate and complete correction was associated with the return of net absorption. Thus, in vivo, partial and complete correction was associated with appropriate changes in vitro in cortical collecting
91
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FIG. 2. The correlation between net chloride balance in vivo and net bicarbonate flux in vitro is shown. Chloride depleted rats demonstrate bicarbonate secretion, correcting rats no net flux, and corrected rats net bicarbonate reabsorption.
duct secretion, if indeed the mechanism of the chloride replacement is as we have hypothesized. We also attempted to induce secretion in vitro by perfusion of tubules in a medium with chloride and bicarbonate concentrations and pH consistent with those of alkalotic blood in vivo, but were unsuccessful in inducing secretion in vitro in these circumstances. Thus, we believe that in vivo events turn off bicarbonate secretion in the CCD during correction of alkalosis but that this mechanism is absent in vitro. It is possibly relevant that ,B agonists stimulate in vitro secretion in the rabbit (4) and that vasointestinal peptide (VIP) stimulates bicarbonate secretion in the turtle bladder which has intercalated cells similar to those in the CCD (31). In summary, we believe that we have demonstrated that chloride is the single and necessary mechanism for the maintenance of CDA and that chloride repletion results in bicarbonate secretion via luminal chloride bicarbonate exchange in the B intercalated cell in the cortical collecting duct. The details on the control mechanisms for chloride bicarbonate exchange remain to be further elucidated. Further support for the possibly important role of chloride bicarbonate exchange in the collecting duct in maintaining systemic acid base balance
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is provided by evidence in man (32) and the rat (personal communication, Wall, Galla and Luke) that sodium bicarbonate loading produces metabolic alkalosis in circumstances of dietary chloride restriction but not when chloride intake is normal. It has also been noted that neonatal rabbits lack fully developed B-cells and have a mild metabolic alkalosis until these B intercalated cells mature (33). The specific effects of alterations in total body chloride and of alterations in chloride delivery rates and transport processes in various segments of the nephron are numerous and still being discovered. Chloridebicarbonate exchange in the collecting duct provides the kidney a mechanism for acid-base adjustments independent of sodium transport and excretion. REFERENCES 1. Aronson PS. Distribution of Sodium Chloride Across Cell Membranes. In Seldin DW, Giebisch G, eds. The Regulation of Sodium and Chloride Balance. New York: Raven; 1990: 3. 2. Koeppen BM. Mechanisms of Segmental Sodium and Chloride Reabsorption. In Seldin DW, Giebisch G, eds. The Regulation of Sodium and Chloride Balance. New York: Raven; 1990: 59. 3. Kirk KL. Defective Regulation of Epithelial Cl- Permeability and Protein Secretion in Cystic Fibrosis: The Putative Basic Defect. Am J Kidney Dis 1989; XIV: 333. 4. Koeppen BM, Giebisch G. Segmental Hydrogen Ion Transport. In Seldin DW, Giebisch G, eds. The Regulation of Acid-Base Balance. New York: Raven; 1989: 139. 5. Wright FS, Briggs JP. Feedback regulation of glomerular filtration rate. Am J Physiol 1977; 233: Fl. 6. Galla JH, Luke RG. Pathophysiology of Metabolic Alkalosis. Hosp Prac 1987: 123. 7. Kurtz TW, Al-Bander HA, Morris RC. "Salt-sensitive" essential hypertension in men. Is the sodium ion alone important? N Engl J Med 1987; 317: 1043. 8. Garella S, Northrup TE, Cohen JJ. Chloride-depletion metabolic alkalosis (CDMA) induces ECF volume (ECFV) contraction via internal fluid shifts in nephrectomized dogs. Kidney Int 1988; 33: 400. 9. Schambelan M, Sebastian A, Rector FC. Mineralocorticoid-resistant renal hyperkalemia without salt wasting (type II pseudohypoaldosteronism): Role of increased renal chloride reabsorption. Kidney Int New York: Raven; 1981; 716. 10. Emmett M, Seldin DW. Clinical syndromes of metabolic acidosis and metabolic alkalosis. In Seldin DW, Giebisch G, eds. The Kidney, Physiology and Pathophysiology. New York: Raven; 1985: 1611. 11. Cohen JJ. Correction of metabolic alkalosis by the kidney after isometric expansion of extracellular fluid. J Clin Invest 1968; 47: 1181. 12. Cogan MG, Liu FY. Metabolic alkalosis in the rat. Evidence that reduced glomerular filtration rate rather than enhanced tubular bicarbonate reabsorption is responsible for maintaining the alkalotic state. J Clin Invest 1983; 71: 1141. 13. Luke RG, Galla JH. Chloride-depletion alkalosis with a normal extracellular fluid volume. Am J Physiol 1983; 245: F419. 14. Galla JH, Bonduris DN, Luke RG. Correction of acute chloride-depletion alkalosis in the rat without volume expansion. Am J Physiol 1983; 244: F217.
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15. Galla JH, Bonduris DN, Dumbauld SL, et al. Segmental chloride and fluid handling during correction of chloride-depletion alkalosis without volume expansion in the rat. J Clin Invest 1984; 73: 96. 16. Galla JH, Bonduris DN, Sanders PW, et al. Volume-independent reductions in glomerular filtration rate in acute chloride-depletion alkalosis in the rat. J Clin Invest 1984; 74: 2002. 17. Galla JH, Bonduris DN, Luke RG. Effects of chloride and extracellular fluid volume on bicarbonate reabsorption along the nephron in metabolic alkalosis in the rat. J Clin Invest 1987; 80: 41. 18. Galla JH, Luke RG. Chloride transport and disorders of acid-base balance. Ann Rev Physiol 1988; 50: 141. 19. Wall BM, Byrum GV, Galla JH, et al. Importance of chloride for the correction of chronic chloride depletion metabolic alkalosis in the rat. Am J Physiol 1987; 253: F1031. 20. Craig DM, Galla JH, Bonduris DN, et al. Importance of the kidney in the correction of chloride-depletion alkalosis in the rat. Am J Physiol 1986; 250: F54. 21. Kassirer JP, Schwartz WB. Correction of metabolic alkalosis in man without repair of potassium deficiency. A re-evaluation of the role of potassium. Am J Med 1966; 40: 19. 22. Rosen RA, Julian BA, Dubovsky EA, et al. On the mechanism by which chloride corrects metabolic alkalosis in man. Am J Med 1988; 84: 449. 23. Kassirer JP, Schwartz WB. The response of normal man to selective depletion of hydrochloric acid. Factors in the genesis of persistent gastric alkalosis. Am J Med 1966; 40: 10. 24. Galla JH, Bonduris DN, Luke RG. Superficial distal and deep nephrons in the correction of metabolic alkalosis. Am J Physiol 1989 257: F107. 25. Atkins JL, Burg MB. Bicarbonate transport by isolated perfused rat collecting ducts. Am J Physiol 1985; 249: F485. 26. Gifford JD, Sharkins K, Work J, et al. Total CO2 transport in rat cortical collecting duct in chloride-depletion alkalosis. Am J Physiol 1990; 258: F848. 27. Verlander JW, Madsen KM, Galla JH, et al. Ultrastructural Localization of H+ATPase in Rat Cortical Collecting Duct (CCD) During Chloride Depletion Metabolic Alkalosis (CDA). JASN 1990; 1: 659. (Abstract) 28. Furuya M, Breyer M, Jacobson H. Functional Characterization of a and : Intercalated Cell Types in the Rabbit Cortical Collecting Duct. JASN 1990; 1: 649. (Abstract) 29. Gifford JD, Galla JH, Luke RG, et al. Ion concentrations in the rat cortical collecting duct: differences between cell types and effect of alkalosis. Am J Physiol 1990; 259: F778. 30. Gifford JD, Sharkins K, Luke RG, et al. Metabolic Alkalosis: in vivo Cl- Repletion Correlates with Cortical Collecting Duct Total CO2 Transport in vitro. JASN 1990; 1: 649. (Abstract) 31. Durham JH, Matons C, Brodsky WA. Vasoactive intestinal peptide stimulates alkali excretion in turtle urinary bladder. Am J Physiol 1987; (Cell Physiol 21) 252: C428. 32. Cogan MG, Carneiro J, Tatsuno J. Normal Diet NaCl Variation Can Affect the Renal Set-Point for Plasma pH-(HCO3) Maintenance. JASN 1990; 1: 193. 33. Satlin LM, Schwartz GJ. Postnatal Maturation of Intercalcated Cells (ICs) in the Rabbit cortical Collecting Duct (CCD). JASN 1990; 1-658. (Abstract)
DISCUSSION Christy (New York): May I exercise my privilege as Chairman to ask you one question if you can answer it simply. I'm afraid maybe you can't, but in the syndrome of so-called
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inappropriate ADH secretion, can you give us some quantitative idea of the waste of chloride vs sodium? Luke: In SIADH sodium and chloride are reduced proportionately mainly because of water retention rather than urinary losses of sodium and chloride. The great interest in the syndrome is why the serum bicarbonate does not fall. Schwartz did some beautiful studies where he showed that aldo levels, in fact, rose ultimately with low sodium and it may be there is a stimulating distal mechanism, that maintains what is a relative alkalosis in SIADH. So I am not answering your question, I am answering a related one that the interesting thing in this syndrome is not the hypochloremia which is proportionate to the sodium, but why does the bicarbonate stay normal. Christy (New York): How about potassium? Luke: The potassium tends to fall a little bit, but remains and falls in proportion, in my experience, to the sodium and chloride. Ferris (Minneapolis): Robin, all us younger Nephrologists accept the fact that chloride is actively handled by the kidneys. It's intriguing though, that with cystic fibrosis, where there is a defect in chloride absorption and so many aberrations in terms of secretions from the lung, there's never been any evidence that their renal physiological mechanism isn't intact. They dilute normally, and their renin secretion seems normal. Luke: The main new idea in cystic fibrosis is that regulation of the epithelial chloride channel in the chloride secreting bronchial apical cell is abnormal. (Kirk KL: Defective regulation of epithelial CL permeability and protein secretion in cystic fibrosis: the putative basic defect. Am J Kidney Dis 1989; 14:333) This causes the viscous bronchial secretions and subsequent respiratory problems and suggests that we may be able to restore normal regulation by drug therapy rather than perhaps having to use gene therapy. Rochester (Charlottesville): As a Pulmonologist, I recall a number of papers from the late 1950's that emphasized that in the recovery from hypercapnia respiratory failure, it was necessary to provide chloride and potassium and the questions that I wanted to ask you first were: in your rat model, does Diamox work if you don't replete chloride? And the other question is when you make your rats alkalotic, do they retain C02? Luke: They retain CO2 and I think that in animals and man CO2 retention in proportion to metabolic alkalosis is unequivocal. I think there is a 0.6 to 0.7 delta increase in CO2 for each milliequivalent increase in serum bicarbonate. I didn't use Diamox in the rats. In my mind, I think you simply compound the disturbance and make a metabolic acidosis as well as a metabolic alkalosis. You're talking about a cor pulmonale patient who is edematous and you can't give him sodium chloride and whose potassium is often high; I think you can give them Diomox and KCL, the two have to be combined. Dunn (Cleveland): That was an elegant summary. There are many parallels between what you described in patients with Bartter's syndrome and yet the putative defective site in Bartter's syndrome is quite different than the site that you focused on for the chloride biocarbonate exchange in the more distal nephron. Would you comment on that? Luke: Bartter's syndrome is not a pure chloride depletion alkalosis but rather is a mixed potassium depletion-aldosterone excess and chloride depletion alkalosis. One might call the syndrome "congenital lasixemia": the NAK2Cl transporter mechanism is likely abnormal in the thick ascending limb. Thus my concept about the Bartter's syndrome is that the collecting duct seems to be doing pretty well and that the defect is in the thick ascending limb which results in increased delivery of sodium potassium and chloride that overwhelms the collecting duct. J. Stein believes, in contrast, that the syndrome may be due to primary K wasting, and indeed there may be several mechanisms resulting in the syndrome. Chloride depletion alkalosis and Bartter's syndrome are clearly different, and Bartter's syndrome is an alkalosis of mixed etiology. Earle (Chicago): This is an old geezer question. Almost 40 years ago, Farber, Pellegrino,
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Alexander and I studied renal function and electrolyte metabolism in twelve episodes of acute glomerulonephritis in adults. We observed a moderate, but significant hyperchloremia. At the time, the patients were edematous, had slightly low serum sodium and were excreting excess amounts of ammonium chloride. This hyperchloremia persisted for weeks or months after everything else except abnormal urine had returned to normal. Could I ask you to explain this now? We had no explanation. Luke: This was hyperchloremic alkalosis. You know classically in chronic renal failure, the attitude has been that you get an anion gap acidosis early on. It's now quite clear that with creatinine of 2, 3, 4 and GFR's perhaps 20-50-60, the first change you get with progressive nephron loss is hyperchloremic, non-anion gap acidosis. The mechanism remains unknown. Now, did your patients have renal insufficiency? Earle (Chicago): Mild to moderate, and transient. Luke: I think that's the answer then. What's in some textbooks at the moment is that chronic renal failure is associated with anion gap acidosis like so many other things in the textbooks that someone else said. This is wrong, and if you really look at it carefully a nonanion gap acidosis is common. Indeed some people, including Jordan Cohen, have postulated that chloride transport is abnormal in some way in early chronic renal failure before bicarbonate transport is abnormal. It remains unknown why non-anion gap hyperchloremic acidosis is the first acidosis acquired by most patients with chronic renal failure. Only later do they get an anion gap acidosis. Earle (Chicago): These were acute. Luke: But you were following them over some weeks weren't you? Earle (Chicago): Yes, that's correct.
