Nongenomic Actions of Aldosterone on the Renal Tubule

HomeHypertensionVol. 49, No. 4Nongenomic Actions of Aldosterone on the Renal Tubule Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessReview ArticlePDF/EPUBNongenomic Actions of Aldosterone on the Renal Tubule David W. Good David W. GoodDavid W. Good From the Departments of Medicine and Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston. Originally published19 Feb 2007https://doi.org/10.1161/01.HYP.0000259797.48382.b2Hypertension. 2007;49:728–739Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: February 19, 2007: Previous Version 1 Aldosterone plays a major role in the maintenance of sodium, potassium, and acid-base balance through its effects on renal electrolyte excretion. This regulation is achieved through aldosterone-induced stimulation of Na+ absorption, K+ secretion, and H+ secretion by the distal nephron, particularly segments of the collecting duct.1–3 These classical actions are mediated through binding of aldosterone to the intracellular mineralocorticoid receptor (MR). The hormone-receptor complex translocates to the nucleus, where it promotes gene transcription and the production of proteins that modulate the expression and activity of the epithelial Na+ channel (ENaC) and other ion transport proteins.1,4–6 Regulatory actions of aldosterone via the MR play an important role in the normal maintenance of blood pressure but also have been implicated in the pathogenesis of hypertension and the progression of renal disease.6–9In addition to their classical actions, aldosterone and other steroid hormones influence cell processes through nongenomic mechanisms.10,11 Nongenomic effects of aldosterone have been demonstrated in many different epithelial and nonepithelial tissues and are defined by (1) an insensitivity to inhibitors of transcription (actinomycin D) and translation (cycloheximide) and (2) a rapid time course (seconds to a few minutes) that is incompatible with gene regulation and de novo protein synthesis. A rapid onset of action is a sufficient but not necessary criterion for a nongenomic effect. Some nongenomic effects can occur with a slower time course. An additional feature often associated with nongenomic effects is that they are not blocked by spironolactone and/or other MR antagonists, consistent with mediation via a nonclassical aldosterone receptor.10,11 Although compelling evidence exists for rapid effects of aldosterone unrelated to the MR, a novel aldosterone receptor for nongenomic regulation has not been identified, and there is evidence that nongenomic actions of aldosterone can be mediated via the classical MR.12 Thus, the role of classical versus nonclassical receptors in mediating nongenomic effects of aldosterone remains controversial. An additional area of uncertainty has been whether nongenomic mechanisms are relevant to aldosterone-induced regulation of renal tubule function, skepticism fueled in part by the fact that most experiments on renal cells have been carried out using cell culture systems. Recently, however, studies using isolated, perfused tubules have established with certainty that aldosterone can regulate the transepithelial transport function of native renal tubules through nongenomic pathways.This review presents a concise update of nongenomic effects of aldosterone on the mammalian renal tubule. Emphasis is placed on ion transport proteins regulated by aldosterone through nongenomic mechanisms, the signal transduction pathways that mediate this regulation, and the possible physiological relevance of nongenomic pathways to the function of nephron segments. Current information is presented with the goal of identifying gaps in knowledge and stimulating future work in this area. The reader is referred to recent reviews for discussion of nongenomic effects of aldosterone on other tissues, including the cardiovascular system.10–14Ion Transport Proteins and Signal Transduction PathwaysA number of ion transport proteins important for the absorptive and secretory functions of renal tubules have been reported to be targets for nongenomic regulation by aldosterone. This section summarizes these transport effects, along with the signal transduction pathways involved (Table). Nongenomic Regulation of Ion Transport Proteins by Aldosterone in Renal CellsTransport ProteinTransport ActivitySignaling Pathway(s)Tubule Segment or Cell LineReferencesNa+/H+ exchanger NHE1↑[Ca2+]i; ERKMDCK-C1124–26 NHE3↓ERKMTAL20,37H+-ATPase↑PKC; microtubulesOMCD38Na+ channel (ENaC)↑MethylationIsolated principal cells (CCD)42Na+-K+ ATPase↑pHi(?)CCD; MDCK48,49Na+/H+ ExchangeAt least 8 mammalian Na+/H+ exchanger isoforms (NHE1 through NHE8) have been identified.15 There is evidence for nongenomic regulation of NHE1 and NHE3 by aldosterone in renal cells. NHE1 is expressed ubiquitously in the plasma membrane of nonpolarized cells and in the basolateral membrane of epithelial cells, where it plays a role in essential functions, such as the regulation of intracellular pH and cell volume, cell growth, and adhesion and migration.15–17 NHE3 is expressed selectively in the apical membrane of certain renal and intestinal epithelial cells, where it mediates transepithelial absorption of Na+ and secretion of H+ necessary for renal HCO3− absorption.3,15,18–20NHE1Stimulation of plasma membrane Na+/H+ exchange was identified as an early response of various renal cells to aldosterone,21–23 but whether this regulation occurred independent of changes in gene expression was not established. Nongenomic stimulation of Na+/H+ exchange by aldosterone was confirmed subsequently in a subtype of Madin-Darby canine kidney cells (MDCK-C11), a cell line that exhibits properties of collecting duct intercalated cells and responds genomically to aldosterone stimulation.24–26 Physiological concentrations (<1 nM) of aldosterone induced rapid (1 to 2 minutes) increases in intracellular pH and Na+ concentration that were attributable to stimulation of Na+/H+ exchange.24–26 These effects were not prevented by actinomycin D, cycloheximide, or spironolactone and were the result of an increase in the affinity of the exchanger for intracellular H+. It is likely that the regulated exchanger is NHE1 based on the following: (1) Na+/H+ exchange stimulated by aldosterone is expressed in the basolateral membrane,26 (2) the inhibitor sensitivity of the exchanger is consistent with that of NHE1,15,16,24,26 and (3) expression of NHE1 was confirmed by immunoblotting in a separate MDCK cell line.27 Aldosterone induces a similar, rapid increase in Na+/H+ exchange activity in a wide variety of epithelial and nonepithelial cells,10,12,28 effects attributed to stimulation of the ubiquitously expressed NHE1 isoform.26,29,30The nongenomic stimulation of NHE1 by aldosterone in MDCK-C11 cells required the parallel activation of a membrane H+ conductance, possibly to permit H+ recycling that prevents the local formation of H+ gradients that could impair Na+/H+ exchange regulation.24,31 Blocking activation of the H+ conductance with Zn2+ or inhibitors of protein kinase C (PKC) prevented the rapid effect of aldosterone to increase Na+/H+ exchange activity. There are no reports in other cell systems that nongenomic stimulation of Na+/H+ exchange by aldosterone depends on the parallel activation of an acid loading mechanism; thus, it is unclear whether this represents a cell-specific property of aldosterone regulation in MDCK-C11 cells.Rapid stimulation of Na+/H+ exchange by aldosterone in MDCK-C11 cells depended on an increase in intracellular Ca2+ concentration due to increased Ca2+ uptake from the extracellular fluid.24 Aldosterone also induced rapid phosphorylation of extracellular signal-regulated kinase (ERK)1/2, and inhibiting ERK1/2 activation prevented the aldosterone-induced increase in Na+/H+ exchange activity.26 Study of the interaction between the 2 signaling pathways showed that preventing the increase in intracellular Ca2+ eliminates the aldosterone-induced stimulation of Na+/H+ exchange but does not prevent increased ERK phosphorylation,24,26,32 whereas blocking ERK activation inhibits the increase in intracellular Ca2+.32 These results suggest that activation of ERK is necessary but not sufficient to stimulate Na+/H+ exchange and that the aldosterone-induced increase in NHE1 activity is mediated through an ERK-dependent increase in intracellular Ca2+. In other cell systems, nongenomic activation of Na+/H+ exchange by aldosterone involves activation of PKC.10 In MDCK-C11 cells, PKC inhibitors prevented the aldosterone-induced increase in Na+/H+ exchange activity, but this was attributed to an indirect effect due to inhibition of the H+ conductance linked to Na+/H+ exchange activity.31NHE1 is expressed in virtually all segments of the renal tubule (see Reference 33 for relevant references). Despite the extensive literature supporting nongenomic regulation of this transporter by aldosterone, there have been no studies testing for an effect of aldosterone on basolateral Na+/H+ exchange in a mammalian nephron segment. Hence, it is unknown whether this prevalent regulatory effect is of significance for renal tubule function. Aldosterone-induced changes in cell pH or Na+ concentration via NHE1 could affect the activity of other renal transport proteins important for ion absorption and secretion or generate a permissive intracellular pH for subsequent nuclear events and protein processing. In addition, NHE1 functions as a signaling molecule to regulate a variety of vital cell functions, including epithelial transport, by controlling the organization of the actin cytoskeleton.16,17,34 Of relevance, basolateral NHE1 has been shown to regulate apical NHE3 and transepithelial HCO3− absorption in the renal medullary thick ascending limb (MTAL) through cytoskeletal remodeling.34 Other transport proteins important for renal function are regulated through cytoskeletal interactions, including ENaC, the renal outer medullary K+ channel (ROMK) potassium channel, and the Na+-K+-2Cl− cotransporter NKCC2 (see References 17 and 34 for relevant references). This suggests that aldosterone could regulate multiple transporters nongenomically through NHE1-induced cytoskeletal reorganization.NHE3Apical NHE3 mediates the majority of NaCl, NaHCO3, and fluid absorption by the renal proximal tubule and virtually all of NaHCO3 absorption by the MTAL.3,15,18–20,34 Regulation of NHE3 is critical for the normal maintenance of extracellular fluid volume, blood pressure, and acid-base balance.18 Recent studies using rat MTALs perfused in vitro have identified NHE3 as a target for nongenomic regulation by aldosterone. Physiological concentrations (IC50=0.6 nM) of aldosterone added to the basolateral solution induced a rapid (<5-minute) inhibition of HCO3− absorption that was the result of a decrease in apical NHE3 activity.20,35 This inhibition was not blocked by inhibitors of transcription or translation or by spironolactone and was not reproduced by glucocorticoids, indicating a high degree of specificity for aldosterone.35 Aldosterone inhibited NHE3 through a reduction in the exchanger's maximal velocity.20 In OKP cells, a cell culture model of renal proximal tubule, glucocorticoids induced an acute increase in maximal velocity of NHE3 that was not blocked by cycloheximide, consistent with nongenomic regulation.36 This effect was the result of glucocorticoid-induced exocytic insertion of NHE3 from intracellular vesicles to the plasma membrane.36 Whether the nongenomic regulation of NHE3 by aldosterone in the MTAL may involve membrane trafficking requires further study.Aldosterone inhibits NHE3 in the MTAL through rapid activation of the ERK1/2 signaling pathway.37 Aldosterone increases ERK activity 2-fold in microdissected MTALs, and blocking ERK activation prevents the effects of aldosterone to inhibit NHE3 and HCO3− absorption.37 The increase in ERK activity is not blocked by actinomycin D or spironolactone. The ERK pathway thus has emerged as an important nongenomic signaling mechanism for aldosterone in the MTAL and collecting duct cells and is a key mediator of aldosterone-induced regulation of both the NHE3 and NHE1 Na+/H+ exchangers.26,37The preceding results suggest that, in addition to its regulation of classical targets such as ENaC and the Na+-K+ ATPase, aldosterone may influence sodium, volume, and acid-base balance through nongenomic regulation of NHE3. Whether aldosterone influences NHE3 activity through nongenomic actions in other epithelial tissues remains to be determined.Vacuolar H+-ATPaseProton secretion via vacuolar H+-ATPases in the collecting duct is the final step in urine acidification and is finely regulated to maintain acid-base homeostasis. Aldosterone influences net acid excretion and acid-base balance through genomic stimulation of the apical H+-ATPase in collecting duct cells.3 A rapid effect of aldosterone on H+-ATPase activity has been described recently. In outer medullary collecting ducts microdissected from normal mice, exposure to 10 nM aldosterone for 15 minutes increased H+ extrusion from acid-loaded, type A intercalated cells.38 This effect was independent of Na+ and blocked by concanamycin, consistent with stimulation of an H+-ATPase. The aldosterone-induced stimulation was partially reduced by actinomycin D but was unaffected by cycloheximide or spironolactone, suggesting a predominantly nongenomic mechanism.38 The stimulation was blocked by the PKC inhibitor chelerythrine Cl and by colchicine, suggesting a dependence on microtubules. In mice injected intraperitoneally with aldosterone for 30 minutes, immunocytochemical analysis showed increased apical staining of the a4 H+-ATPase subunit in type A intercalated cells.38 These results suggest that the rapid action of aldosterone involves increased trafficking of H+-ATPase to the apical membrane.Some aspects of the preceding study limit conclusions regarding the physiological significance of the results. First, the stimulation of pump activity was not observed with 1 nM aldosterone, a physiological concentration that typically induces nongenomic effects. Second, the partial block of H+-ATPase stimulation by actinomycin D raises uncertainty as to whether the length of treatment with this inhibitor (15 minutes) was sufficient to fully inhibit gene transcription. Third, the transport studies were carried out on unperfused tubules. Thus, it remains to be determined whether the rapid increase in cellular H+ extrusion is associated with a physiologically relevant change in transcellular H+ secretion. Despite these issues, the results provide important evidence for rapid regulation of ion transport by aldosterone in native renal tubules and identify the vacuolar H+-ATPase in intercalated cells as a focal point for future studies of nongenomic regulation.ENaCENaC is located in the apical membrane of principal cells in the connecting tubule and collecting duct, where it mediates transepithelial Na+ absorption.2,4,7 The regulation of ENaC is critical for the maintenance of Na+ and volume balance and normal blood pressure.6,7 Aldosterone stimulates Na+ absorption in collecting ducts by increasing ENaC activity. This occurs through changes in gene transcription via the classical MR, resulting in the induction of regulatory proteins, such as SGK1, that lead to an increase in the activity and number of functional channels in the apical membrane.4–7,39–41 In addition to this classical regulation, there is evidence for rapid activation of ENaC by aldosterone through nongenomic mechanisms. Na+ channel activity was assessed using whole cell patch clamp techniques in principal cells freshly isolated from rabbit cortical collecting ducts (CCDs).42 Exposure to 100 nM aldosterone increased the activity of a highly selective, amiloride-sensitive Na+ conductance within 1 to 2 minutes.42 This effect was not prevented by spironolactone but was blocked by S-adenosyl-l-homocysteine, suggesting that transmethylation reactions may play a role in nongenomic, as well as early genomic,43 regulation of ENaC by aldosterone.Caution is warranted with respect to the physiological significance of the above findings for collecting duct function. The rapid stimulation of ENaC was reported only for a pharmacological concentration of aldosterone, and specificity of the Na+ channel stimulation for aldosterone was not established (glucocorticoids were not tested). Also, aldosterone did not stimulate Na+ channel activity in principal cells isolated from CCDs of rats.42 In previous studies examining the acute in vitro effects of aldosterone in the isolated, perfused CCD, a rapid stimulation of Na+ absorption has not been reported.44–46 It should be noted, however, that these studies were not designed to analyze Na+ transport immediately after aldosterone addition, and measurements of absorptive Na+ flux were not reported until 30 to 120 minutes after bath aldosterone addition. Based on the patch clamp data for ENaC in principal cells, further work is needed to assess whether nongenomic pathways are relevant to aldosterone-induced stimulation of collecting duct Na+ absorption.Na+-K+ ATPaseIn addition to stimulating ENaC, aldosterone increases Na+-K+ ATPase activity through transcriptional regulation in collecting duct principal cells,1,4,47 resulting in a coordinated regulation of apical and basolateral membrane transport proteins that promotes an efficient increase in transcellular Na+ absorption. Two studies have implicated a possible role for nongenomic pathways in aldosterone-induced Na+ pump regulation in renal cells. In CCDs microdissected from adrenal-intact rats, aldosterone increased ouabain-sensitive 86Rb uptake within 30 minutes at a half-maximal concentration of 1 nM.48 This effect was partially reduced by actinomycin D and cycloheximide; however, ≈30% of the stimulation of pump activity persisted in the presence of the 2 inhibitors.48 In MDCK cells, aldosterone at 10−8 to 10−6 M increased Na+ pump activity at 15 minutes with a plateau at 60 minutes, as assessed under maximal velocity conditions by a cytochemical assay and specific 3[H]ouabain binding.49 The increase in activity was blocked by colchicine but was unaffected by cycloheximide over 30 minutes. In both MDCK cells and CCDs, the stimulation of Na+ pump activity was unchanged in the presence of ionophores to increase membrane Na+ permeability.48,49 Hence, the increase in Na+-K+ ATPase activity is unlikely to be the indirect result of increased apical Na+ uptake via aldosterone-induced ENaC activation. On the other hand, in MDCK cells, pump activation by aldosterone was prevented by pretreatment with dimethylamiloride, an inhibitor of Na+/H+ exchange.49 This raises the possibility that nongenomic stimulation of Na+-K+ ATPase in collecting duct cells depends on Na+/H+ exchange, whereby aldosterone induces a rapid increase in NHE1 activity,26 resulting in a rise in intracellular pH that secondarily increases Na+-K+ ATPase activity.50 Thus, although nongenomic regulation of Na+-K+ ATPase activity by aldosterone appears likely in renal cells, further work is needed to determine whether this involves direct or indirect coupling of nongenomic pathways to pump activation.OtherAldosterone influences renal Na+ absorption through regulation of the thiazide-sensitive Na-Cl cotransporter in the distal convoluted tubule,51,52 but the possible role of nongenomic mechanisms is unknown. Aldosterone regulates the Na+-K+-2Cl− cotransporter NKCC1 in cardiac myocytes through nongenomic mechanisms.14 NKCC1 is expressed in the connecting tubule, collecting duct, and juxtaglomerular apparatus, where it participates in processes such as Cl− secretion, renin secretion, and tubuloglomerular feedback (see References 53 and 54 and references therein). Whether aldosterone influences NKCC1 in the kidney has not been explored. Finally, aldosterone affects a number of signaling pathways in renal cells in addition to those listed in the Table, including cAMP/protein kinase A (PKA),55,56 calcineurin,57 Src,58 and heat shock proteins.57,58 Their possible significance for nephron function is considered below.Function of Defined Nephron Segments and Whole KidneyStudies using isolated nephron segments and segment-derived cell lines have identified the MTAL, segments of the collecting duct, and possibly the proximal tubule as sites of nongenomic regulation by aldosterone. Findings relevant to specific nephron segments and their potential significance for tubule function are presented briefly below. Rapid effects of aldosterone on whole kidney function in vivo also are summarized.MTALThe MTAL has been identified conclusively as a target for nongenomic regulation, with aldosterone inducing rapid inhibition of apical NHE3 and HCO3− absorption.20,35 Although inhibition of NHE3 appears counterintuitive for the physiological function of aldosterone to promote renal Na+ retention, this effect in the MTAL may serve an important function for acid-base homeostasis. In the proximal tubule, NHE3 is the primary mediator of Na+ absorption, both as NaCl and NaHCO3.3,18,19 In the MTAL, NHE3 mediates absorption of NaHCO3 (see References 20 and 34 for relevant references), but absorption of NaCl, which accounts for the majority of Na+ absorption, is mediated by NKCC2.59,60 Thus, in the MTAL, changes in NHE3 activity are important for HCO3− absorption and acid-base balance but have little impact on net Na+ absorption.59 Within this context, the inhibition of NHE3 by aldosterone in the MTAL may play a key role in enabling the kidney to regulate Na+ balance and extracellular fluid volume while maintaining acid-base balance. Activation of the renin-angiotensin-aldosterone system by sodium and volume depletion promotes renal Na+ retention but also induces multiple transport effects that tend to increase renal net acid excretion and promote metabolic alkalosis. The latter includes stimulation of HCO3− absorption by angiotensin II in segments of the proximal and distal tubule, stimulation of ammonium secretion by angiotensin II in the proximal tubule, and stimulation of H+ secretion by aldosterone in segments of the collecting duct (see Reference 35 for relevant references). The direct action of aldosterone to inhibit H+ secretion and HCO3− absorption in the MTAL would oppose these changes in the proximal and distal tubules. In this way, nongenomic regulation of NHE3 by aldosterone in the MTAL would tend to minimize changes in H+ excretion and maintain acid-base balance while permitting regulated changes in Na+ excretion that are necessary for regulation of extracellular fluid volume and blood pressure. This counterregulatory function of the MTAL is magnified further by the concurrent inhibition of HCO3− absorption by angiotensin II, which is additive to inhibition by aldosterone.35 Consistent with this view, rapid infusion of aldosterone into intact rats induced a significant change in renal Na+ excretion with no effect on urinary pH or acid excretion.61 On the other hand, the nongenomic inhibition of HCO3− absorption in the MTAL may contribute to acid-base disorders associated with changes in systemic potassium balance. For example, an increase in NHE3 activity and HCO3− absorption in the MTAL in response to a reduced plasma aldosterone level could contribute to the increased renal HCO3− absorptive capacity that promotes metabolic alkalosis in K+ depletion.3,35Absorption of NaCl by the MTAL is important for the maintenance of Na+ balance and blood pressure and for the excretion of a concentrated or dilute urine.7,60,62 NaCl absorption depends on apical NaCl uptake via NKCC2, apical K+ recycling via ROMK, and basolateral Cl− via Cl− It is unknown whether these transport proteins rapid regulation by aldosterone or they are regulated by the ERK signaling which is by aldosterone in the aldosterone has been shown to a rapid increase in in a distal cell line isolated by using a of thick ascending limb this effect is confirmed for native MTALs, aldosterone could stimulate NaCl absorption nongenomically via CCD, outer medullary collecting duct, and medullary collecting duct have been identified as sites of nongenomic regulation by aldosterone. Although a nongenomic action of aldosterone to regulate transepithelial transport in these segments has not been key for future can be aldosterone increases H+-ATPase activity in the outer medullary collecting and ENaC activity in principal cells isolated from rabbit suggesting that nongenomic pathways may contribute to the physiological actions of aldosterone to stimulate H+ secretion and Na+ absorption by collecting duct segments. The latter possibility is by the in the cell line that aldosterone induces an early increase in that is not blocked by actinomycin D or that ENaC, ROMK, and the Na+-K+ ATPase can be by cell aldosterone could influence Na+ absorption or K+ secretion in the collecting duct by increasing cell pH through nongenomic stimulation of This has been but has to be in mammalian collecting duct respect to signaling aldosterone induces a rapid increase in ERK activity in both and collecting duct cell Although ERK appears to play a role in the regulation of Na+ transport by aldosterone, this role is In a cell line from principal cells inhibition of ERK under both and effects attributed to a decrease in Na+-K+ ATPase This suggests that the early activation of ERK by aldosterone may the stimulation of Na+ absorption through a permissive role that the function of the basolateral Na+ On the other hand, activation of ERK by aldosterone or other over a to of ENaC by its interaction with which results in channel and Thus, ERK may be a of a feedback mechanism for of ENaC after aldosterone Hence, nongenomic, and genomic activation of ERK may different regulatory for Na+ absorption. In microdissected rat CCDs, aldosterone induced a increase in the activity of calcineurin,57 which has been implicated in the regulation of Na+-K+ ATPase but of significant aldosterone at physiological concentrations a rapid to increase in intracellular in microdissected rat medullary collecting This increase occurred independent of the and receptors and was additive to increases in induced by These findings the possibility that aldosterone may via nongenomic mechanisms to regulate a number of collecting duct functions, such as absorption via channels and absorption via information is on whether aldosterone may influence proximal tubule function through nongenomic mechanisms. In of proximal tubule cells from renal aldosterone induced a rapid increase in intracellular and a increase in intracellular Ca2+ The Ca2+ response was not blocked by spironolactone and could not be reproduced with Whether this regulation occurs in native proximal tubules is however, and Ca2+ are important of ion transport and the absorptive functions of proximal tubule segments. Aldosterone has been shown to increase the apical and activity of NHE3 in proximal tubules of rats in vivo and in a proximal tubule cell however, these effects are to and the possible role of nongenomic mechanisms was not with the additional that NHE3 is regulated via a nongenomic pathway by glucocorticoids in a proximal cell line and that NHE3 is a target for nongenomic regulation by aldosterone in the there exists a for the that aldosterone may influence proximal Na+ and fluid absorption through nongenomic regulation of rapid effect of aldosterone on the kidney was described as early as by and of aldosterone into the or renal of Na+ excretion and increased K+ excretion within 15 minutes or consistent with a nongenomic Thus, in this the rapid actions of aldosterone with its classical genomic actions to promote Na+ retention and K+ excretion. In in recent studies of rats, infusion of aldosterone induced an increase in Na+ excretion within 15 minutes, with no change in or HCO3− excretion or urine The significance