JOURNAL CLUB GLUCOCORTICOIDS have an important role in the physiological response to systemic acidosis. This role is not only important from a physiol

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1 JOURNAL CLUB GLUCOCORTICOIDS have an important role in the physiological response to systemic acidosis. This role is not only important from a physiological perspective, but also has important clinical implications. However, this interaction of glucocorticoids with acidosis has not been fully appreciated, except by a few groups. In the article under discussion, Glucocorticoids Enhance Acid Activation of the Na /H Exchanger 3 (NHE3), Ambühletal 1 explored the molecular mechanisms of this interaction in the proximal tubule. Adrenal insufficiency is often accompanied by metabolic acidosis, a result of decreased excretion of titratable acid and ammonium. 2,3 Conversely, Cushing s syndrome frequently causes metabolic alkalosis. Although the effects of both these conditions have been attributed in part to distal tubule effects by mineralocorticoid receptors, glucocorticoids have important effects in the proximal tubule, particularly in response to acidosis. In adrenalectomized animals, glucocorticoids increase net acid excretion (ammonium plus titratable acid) more than aldosterone, despite a larger effect of the latter on reducing urine ph. 4 Importantly, in healthy animals or persons, metabolic acidosis results in a corticotropindependent increase in corticosteroids. 2,5,6 In turn, the increase in glucocorticoids with acidosis is necessary for many of the physiological responses to acidosis: increased ammonium excretion, increased titratable acid, and increased proximal tubule sodium-hydrogen (Na /H ) exchange. In a pivotal study, Kinsella et al 7 showed that adrenalectomized animals failed to respond appropriately to metabolic acid loads in terms of ammonium and phosphate excretion; further- From the Section of Nephrology, Tulane University Medical Center, New Orleans, LA. Received and accepted as submitted May 24, Address reprint requests to L. Lee Hamm, MD, Chief, Section of Nephrology, SL 45, Tulane University Medical Center, 1430 Tulane Ave, New Orleans, LA lhamm@tmcpop.tmc.tulane.edu 1999 by the National Kidney Foundation, Inc /99/ $3.00/0 Role of Glucocorticoids in Acidosis Discussion: L. Lee Hamm, MD This month s discussion... The Journal Club focuses on an article entitled Glucocorticoids Enhance Acid Activation of the Na /H Exchanger 3 (NHE3) (J Clin Invest 103: , 1999) by Patrice M. Ambühl, Xiaojing Yang, Yan Peng, Patricia A. Preisig, Orson W. Moe, and Robert J. Alpern. more, this failure could be corrected by the administration of the synthetic glucocorticoid dexamethasone, without replacement of mineralocorticoids. 7 Parallel findings were made for Na /H exchange in brush-border membrane vesicles: increased exchange with vesicles from acidotic animals with intact adrenals, no adaptation with vesicles from adrenalectomized animals even with acidosis, and restoration of the response to acidosis with dexamethasone treatment. 7 The cellular and molecular mechanisms of the NHE findings have now been characterized by Ambühletal, 1 as discussed next. Of note, Na /H exchange and phosphate excretion in the studies of Kinsella et al 7 increased just as much with dexamethasone in nonacidotic as in acidotic animals (or their membrane vesicles). Kinsella et al 7 proposed that glucocorticoids orchestrate the proximal tubule response to acidosis. The changes in ammonium excretion and Na /H exchange previously discussed may be linked, at least in part. Ammonium is produced predominantly in the proximal tubule from glutamine and is secreted into the lumen probably by two mechanisms: (1) Na -NH 4 exchange on the NHE, and (2) NH 3 diffusion with trapping of NH 4 as the lumen ph decreases with Na /H exchange and sodium-independent acidification through H -adenosine triphosphatase. 8 In other words, both mechanisms of ammonium secretion into the lumen depend on the apical membrane NHE. However, there are also other reasons that ammonium secretion is inadequate in the absence of glucocorticoids. Welbourne 6,9 and others have shown that glucocorticoids enable acido- 960 American Journal of Kidney Diseases, Vol 34, No 5 (November), 1999: pp

2 JOURNAL CLUB 961 sis to appropriately increase glutamine uptake, mitochondrial glutaminase, and total ammonium production. Increased glucocorticoids alone, without primary acidosis, increase endogenous acid production (EAP) and decrease proximal tubule phosphate reabsorption The increased EAP may result from decreased protein synthesis and possibly from increased muscle catabolism, as discussed next. Urinary net acid excretion increases, partly in response to the increased EAP, but it eventually may exceed EAP, resulting in elevated plasma bicarbonate levels. 10,11 The decreased phosphate reabsorption with glucocorticoids results from inhibition of the apical membrane sodium-dependent phosphate transporter 13 ; this will allow increased titratable acid excretion because phosphate is the main urinary buffer. More importantly quantitatively, ammonium excretion also increases. Another consequence of systemic acidosis also requires glucocorticoids: muscle breakdown. Although increased muscle catabolism in response to acidosis has been known for many years, recently, Mitch, Price, England, and their colleagues have shown a critical role of glucocorticoids in permitting the effects of acidosis (eg, 14,15 ). In the degradative pathway of muscle proteins, the synergistic interactions of acidosis and glucocorticoids occur in regulating the messenger RNA (mrna) levels of several key enzymes (eg, 15 ), a contrast with the results discussed below. Another potential site of interaction of acidosis with glucocorticoids is bone. In this case, there is no evidence that the effects of acidosis on bone are dependent on glucocorticoids or vice versa. However, both systemic acidosis and glucocorticoids cause increased bone resorption and increased urinary calcium; therefore, the combined effect is likely to be at least additive. In the article under discussion, Ambühletal 1 used Opossum Kidney, Pelone (OKP) cells to address the mechanisms of the interaction of glucocorticoids with acidosis to stimulate proximal tubule Na /H exchange. OKP cells are an opossum proximal tubule cell line that expresses apical membrane NHE3 as in the intact proximal tubule. NHE3 has now been well established as the chief isoform of Na /H exchangers responsible for most of proximal tubule HCO 3 reabsorption. The OKP cell line has been particularly useful to study a variety of proximal tubule transport processes. Ambühletal 1 used a variety of functional and biochemical techniques in fairly standard methods. In these cells, they found that either in vitro acidosis (ph 6.8 versus 7.4) or in vitro hydrocortisone (HC; 10 7 to 10 8 mol/l) increased Na /H exchange, as measured by recovery of intracellular ph from an acid load. The combination was additive (or perhaps more). However, a dose of HC (10 9 ) that had no independent effect also augmented the effect of the acid media. Control experiments showed that neither aldosterone nor an inhibitor of NHE1 had an effect on Na /H exchange. Further experiments by Ambühl et al 1 addressed the molecular mechanisms of these functional effects. Both acidosis and glucocorticoids have been previously shown to increase NHE3 mrna and protein levels. In the present studies, 10 9 mol/l of HC with normal ph media increased both mrna and protein abundance of NHE3, despite the lack of functional effects in the absence of acidosis. Somewhat surprisingly, this low concentration of HC had additive or synergistic activity with acidosis on NHE3 protein synthesis, but not on mrna levels. Importantly, Ambühletal 1 additionally showed that 10 9 mol/l of HC enabled acidosis to increase Na /H exchange in the absence of new protein synthesis (cycloheximide present), a posttranslational effect. At least part of this posttranslational effect appeared to be redistribution of NHE3 protein to the apical membrane (determined by biotinylation of apical membrane proteins). These new studies by Ambühletal 1 clearly show the synergistic interaction of glucocorticoids and acidosis to regulate proximal tubule Na /H exchange. Importantly, they show that this interaction occurs at multiple levels: protein abundance, apical membrane insertion (or retrieval) of protein, and function (Fig 1). These findings present an unexpected paradigm: multiple levels of interaction in a single functional pathway. However, a multilevel interaction is reminiscent of the multiple consequences of mineralocorticoids in the distal nephron and of the effects of glucocorticoids in muscle, as previously described. The studies by Ambühletal 1 were obviously carefully and thoughtfully performed, and the results are important in providing new molecular

3 962 JOURNAL CLUB Fig 1. Schematic of interaction of glucocorticoids and acidosis (see text). Bold arrows indicate sites of apparent synergy. mechanisms of the interaction of glucocorticoids and acidosis. However, as with any report, it is important to ask not only what is new or important, but also, what is wrong. Most articles, even in the most prestigious journals, have some potential flaws, residual uncertainties, or unanswered questions. For instance, the variability of baseline Na /H exchange from experiment to experiment (compare Figs 2 and 5 1 ) provides one note of caution; unfortunately, this is problematic in many cultured cell systems. Some relatively minor additional details would have helped further define the relationship between acidosis and glucocorticoids. Does HC at higher concentrations have effects on antiporter activity in the absence of protein synthesis? Does HC alone without acidosis or acidosis alone without HC increase apical membrane NHE3 abundance? Why do acidosis and glucocorticoids not have at least additive effects on NHE3 mrna levels? Despite these minor issues, the studies appear solid and extensive. As with any important new findings, a variety of new directions are also provided. For instance, what is the mechanism of increased insertion into the apical membrane? Because the activities of the apical NHE and the basolateral sodium bicarbonate transporter frequently change in parallel do glucocorticoids have similar effects on the basolateral sodium bicarbonate transporter? Are there similar multiple levels of interactions between acidosis and glucocorticoids in other physiological responses (eg, ammonia excretion)? Important regulatory processes for NHE3 have recently been shown (direct phosphorylation and interaction with regulatory proteins); are these mechanisms operative in the synergism of acidosis and glucocorticoids? Are other paracrine hormone systems activated? To some degree, the new questions raised by this study illustrate its importance. From a clinical perspective, the present study emphasizes the necessity for glucocorticoids in the normal response to acidosis; glucocorticoiddeficient patients will be unable to appropriately respond to or correct metabolic acidosis or acid loads. The interactions of glucocorticoids and acidosis occur not only in the kidney, but also in muscle and bone, in complex mechanisms. In understanding acid-base homeostasis and derangements of such, both mineralocorticoids and glucocorticoids are important considerations. The article by Ambühletal 1 contributes valuable new information to this understanding. REFERENCES 1. Ambühl P, Yang X, Peng Y, Preisig P, Moe O, Alpern R: Glucocorticoids enhance acid activation of the Na /H exchanger 3 (NHE3). J Clin Invest 103: , Sartorius O, Calhoon D, Pitts R: Studies on the interrelationships of the adrenal cortex and renal ammonia excretion by the rat. Endocrinology 52: , Dubrovsky A, Nair R, Byers M, Levine D: Renal net acid excretion in the adrenalectomized rat. Kidney Int 19: , Wilcox C, Cemerikic D, Giebisch G: Differential effects of acute mineralo- and glucocorticosteroid administration on renal acid elimination. Kidney Int 21: , Perez G, Oster J, Katz F, Vaamonde C: The effect of acute metabolic acidosis on plasma cortisol, renin activity and aldosterone. Hormone Res 11:12-21, Welbourne T: Acidosis activation of the pituitaryadrenal-renal glutaminase I axis. Endocrinology 99: , Kinsella J, Cujdik T, Sacktor B: Na /H exchange activity in renal brush border membrane vesicles in response to metabolic acidosis: The role of glucocorticoids. Proc Natl Acad Sci U S A 81: , 1984

4 JOURNAL CLUB Hamm L, Simon E: Ammonia transport in the proximal tubule. Miner Electrolyte Metab 16: , Welbourne T: Glucocorticoid control of ammoniagenesis in the proximal tubule. Semin Nephrol 10: , Hulter H, Licht J, Bonner E, Glynn R, Sebastian A: Effects of glucocorticoid steroids on renal and systemic acid-base metabolism. Am J Physiol 239:F30-F43, Hulter H, Sigala J, Sebastian A: Effects of dexamethasone on renal and systemic acid-base metabolism. Kidney Int 20:43-49, Anderson J, Foster J: The effect of cortisone on urinary phosphate excretion in man. Clin Sci 18: , Freiberg J, Kinsella J, Sacktor B: Glucocorticoids increase the Na /H exchange and decrease the Na gradientdependent phosphate-uptake systems in renal brush border membrane vesicles. Proc Natl Acad Sci U S A 79: , May R, Kelly R, Mitch W: Metabolic acidosis stimulates protein degradation in rat muscle by a glucocorticoiddependent mechanism. J Clin Invest 77: , Isozaki Y, Mitch W, England B, Price S: Protein degradation and increased mrna encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC 3 H1 myocytes require an interaction between glucocorticoids and acidification. Proc Natl Acad Sci U S A 93: , 1996 Authors Reply: Patrice M. Ambühl, MD, and Robert J. Alpern, MD IN REAL LIFE, as well as in science, new discoveries sometimes are fostered by unanticipated problems, with their subsequent solutions. It had been known for some time that acidosis activates the renal cortical Na /H antiporter (NHE) 1 and that it increases serum glucocorticoid levels in animals and in humans. Dr Hamm has comprehensively reviewed the relevant literature on this topic. Based on the study by Kinsella et al 2 15 years ago, a link between renal Na /H antiporter activation and corticosteroid hormones became apparent in metabolic acidosis. Our own work on the interrelationship between acid and steroids in the regulation of the renal Na /H exchanger, which led to the publication under discussion in this Journal Club, actually began with other experiments on the effect of metabolic acidosis on the renal Na /H antiporter in metabolic acidosis. In our studies published in the American Journal of Physiology, 3 we found that it is the proximal tubule apical membrane Na /H antiporter encoded by the Na /H exchanger 3 (NHE3) that is regulated by metabolic acidosis in rats. Animals fed a diet containing ammonium chloride developed metabolic acidosis within 3 days and showed 20%, 60%, and 100% increases in renal cortical apical membrane NHE3 protein abundance compared with control animals after 3, 7, and 14 days, respectively. This increase was accompanied by a gradual normalization of the acid-base status and occurred in the absence of an increase in NHE3 messenger RNA (mrna) abundance. Based on these results, we intended to further characterize the mechanisms involved in the regulation of NHE3 by acidosis. As stated by Dr Hamm, OKP (Opossum Kidney, Pelone) cells are a valuable tool for the study of NHE3 because this cell line shows many proximal tubular features. NHE3 is regulated similarly in OKP cells and in the intact proximal tubule. Of note, we have shown that incubation of OKP cells in acid media (ph 6.8) causes an increase in NHE3 activity and mrna abundance. 4 Similarly, treatment of OKP cells with dexamethasone increases NHE3 activity and mrna abundance. 5 The present studies began with our attempt to show that acid incubation of OKP cells caused an increase in NHE3 protein abundance. Given that acid incubation increased NHE3 activity and mrna abundance in OKP cells, it seemed almost predictable that protein abundance would also be increased, as seen in the intact proximal tubule in vivo. However, as is typical for easy, predictable experiments, we were not only surprised, but also deeply disappointed to find a minimal effect of acid incubation on OKP NHE3 From the Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX. Received and accepted as submitted July 1, Address reprint requests to Robert J. Alpern, MD, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX robert.alpern@ .swmed.edu 1999 by the National Kidney Foundation, Inc /99/ $3.00/0

5 964 JOURNAL CLUB protein abundance. Indicative of our surprise, this experiment was performed 18 times (see Fig 7 of the article). Even at a ph of 6.8, less than that achieved in vivo in acid-fed rats (minimum ph, 7.2), only a marginal increase in OKP NHE3 protein abundance was obtained. This occurred despite the fact that NHE3 mrna abundance was increased in cultured cells by acid incubation but was not increased in vivo by acidosis. Obviously, intact animals and isolated cultured cells are different in many respects, which may have accounted for the small effect in OKP cells. Although there is a tendency to dismiss such unexpected findings in culture as caused by this myriad of differences, we attempted to consider physiological differences between the preparations as a possible cause. Identifying such a key difference could elucidate an important component of the acid response. Known regulators of the renal antiporter, such as hemodynamic effects and glomerular filtration, cannot be mimicked in cell culture and could be required. Another possibility was a key hormone missing from the culture system. To achieve maximal differentiation in culture, cells are serum deprived for at least 24 hours before and during the time they undergo study. We realize that in doing so, we also deprive our cells of serum components that are implicated in the regulation of the renal Na /H exchanger. The most prominent among these are glucocorticoid hormones. It was at this time that the more junior of us (P.M.A.) was reading the report by Kinsella et al 2 previously mentioned. Our own observations, together with the literature suggesting an interaction of acid(osis) and corticosteroids in regulating renal Na /H exchange, led to the hypothesis that the lack of corticosteroid hormones was responsible for the missing acid effect on OKP NHE3 protein abundance. Indeed, as summarized by Hamm, it turned out that the combination of acid and the glucocorticoid effect of hydrocortisone regulates NHE3 activity in OKP cells by complex mechanisms involving posttranscriptional effects on NHE3 protein. Based on our results, the following conclusions can be made. In cultured OKP cells studied in the absence of exogenous glucocorticoids, acid incubation causes a twofold increase in NHE3 mrna abundance, a 50% increase in NHE3 protein abundance, and an increase in NHE3 activity. When glucocorticoids are added, acid incubation causes the same twofold increase in NHE3 mrna abundance but now causes a threefold greater increase in NHE3 protein abundance and a twofold greater increase in NHE3 activity. The enhanced increase in NHE3 protein abundance appears to be caused by an increase in NHE3 protein synthesis. In addition, these studies suggested that glucocorticoids enhance an acidinduced posttranslational effect on NHE3 trafficking to the apical membrane. The relatively larger effects on NHE3 protein abundance and activity compared with mrna abundance are more consistent with that seen in the in vivo setting. In the intact animal, smaller changes in ph may be insufficient to lead to measurable changes in NHE3 mrna abundance but, in the presence of stimulated cortisol secretion, may be sufficient to elicit increased NHE3 protein synthesis and trafficking to the apical membrane. We would suggest that the reader could learn a number of lessons from these studies. Cell culture can be an important tool to increase our understanding of in vivo phenomena. It is most useful when it recreates an in vivo effect and then allows one to explore the mechanism in a controlled setting. It is also useful when it recreates an in vivo effect, but components of the response differ from the response in vivo. Although Kinsella et al 2 clearly were the first to suggest a role of corticosteroids in the response to acidosis, it was not clear from their study whether glucocorticoids were permissive to renal antiporter regulation by acidosis or whether this was merely a secondary phenomenon caused by the hemodynamic effects of dexamethasone and the consequence of increased glomerular filtration rate. 6 Whereas the in vivo setting is most likely to imitate real life, it is limited by difficulty in controlling all variables or even identifying all variables for which one needs to control. Moreover, in our experiments with OKP cells, we were able to work out subtle conditions that allowed us to differentiate between additive and synergistic effects of glucocorticoids on acidinduced Na /H exchange activation. Nevertheless, several problems with cell culture studies remain, as correctly stated by Hamm. One inherent problem with these experiments is

6 JOURNAL CLUB 965 the variability of baseline Na /H exchange activity. For reasons that are not clear, cells in culture vary from passage to passage, possibly related to their level of confluence, level of differentiation, density, or other unknown factors. It has been our experience that this leads to variability in baseline Na /H antiporter rates but does not alter whether a given determinant increases or decreases activity. For this reason, all comparisons are made between cells from the same passage studied on the same day. It is intrinsic to research that every new finding raises at least as many unanswered questions and provides thought for additional experiments. From our study, as well as the present discussion of our results, it is apparent that the mechanisms by which NHE3 activity is regulated by the interaction of acid(osis) and corticosteroids are multifactorial and require further investigation. It is the integration of clinical observation, animal experiments, and cell culture results that provides us with a better understanding of how the kidney compensates for metabolic acidosis. Glucocorticoids, it seems, are an important player on this stage. REFERENCES 1. Preisig PA, Alpern RJ: Chronic metabolic acidosis causes an adaptation in the apical membrane Na/H antiporter and basolateral membrane Na(HCO 3 ) 3 symporter in the rat proximal convoluted tubule. J Clin Invest 82: , Kinsella J, Cujdik T, Sacktor B: Na /H exchange activity in renal brush border membrane vesicles in response to metabolic acidosis: The role of glucocorticoids. Proc Natl Acad Sci U S A 81: , Ambühl PM, Amemiya-M, Danczkay M, Lötscher M, Kaissling B, Moe OW, Preisig PA, Alpern RJ: Chronic metabolic acidosis increases NHE3 protein abundance in rat kidney. Am J Physiol 271:F917-F925, Amemiya M, Yamaji Y, Cano A, Moe OW, Alpern RJ: Acid incubation increases NHE-3 mrna abundance in OKP cells. Am J Physiol 269:C126-C133, Baum M, Amemiya M, Dwarakanath V, Alpern RJ, Moe OW: Glucocorticoids regulate NHE-3 transcription in OKP cells. Am J Physiol 270: F164-F169, Maddox DA, Fortin SM, Tartini A, Barnes WD, Gennari FJ: Effect of acute changes in glomerular filtration rate on Na /H exchange in rat renal cortex. J Clin Invest 89: , 1992