Regulation of the Cl–/HCO3– Exchanger AE2 in Rat Thick Ascending Limb of Henle’s Loop in Response to Changes in Acid-Base and Sodium Balance
Fabienne Quentin*,
Dominique Eladari*,,
Sebastian Frische,,
Michèle Cambillau||,
Søren Nielsen,,
Seth L. Alper¶,
Michel Paillard*, and
Régine Chambrey*
*Institut National de la Santé et de la Recherche Médicale Unité 356, Institut Fédératif de Recherche 58, Université Paris V, Paris, France; Département de Physiologie, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France; The Water and Salt Research Center, University of Aarhus, Aarhus C, Denmark; Institute of Anatomy, University of Aarhus, Aarhus C, Denmark; ||Service de Biochimie, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France; and ¶Vascular and Molecular Medicine Unit and Renal Unit, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
Correspondence to Dr. Régine Chambrey, Unité INSERM 356, Institut de Recherche des Cordeliers, 15 rue de l’Ecole de Médecine, 75270 Paris Cedex 06, France. Phone: +33-1-44-41-37-10; Fax: +33-1-44-41-37-17; E-mail: chambrey{at}ccr.jussieu.fr
The Cl–/HCO3– exchanger AE2 is believed to be involvedin transcellular bicarbonate reabsorption that occurs in thethick ascending limb of Henle’s loop (TAL). The purposeof this study was to test whether chronic changes in acid-basestatus and sodium intake regulate AE2 polypeptide abundancein the TAL of the rat. Rats were subjected to 6 d of loadingwith NaCl, NH4Cl, NaHCO3, KCl, or KHCO3. AE2 protein abundancewas estimated by semiquantitative immunoblotting in renal membranefractions isolated from the cortex and the outer medulla oftreated and control rats. In the renal cortex, AE2 abundancewas markedly increased in response to oral loading with NH4Clor with NaCl. In contrast, AE2 abundance was unchanged in responseto loading with KCl or with NaHCO3 and was decreased by loadingwith KHCO3. The response of AE2 in the outer medulla differedfrom that in the cortex in that HCO3– loading increasedAE2 abundance when administered with Na+ but had no effect whenadministered with K+. Immunohistochemistry revealed that NaClloading increased AE2 abundance in the basolateral membraneof both the cortical and the medullary TAL. In contrast, NH4Clloading increased AE2 abundance only in the cortical TAL butnot in the medullary TAL. These results suggest that regulationof the basolateral Cl–/HCO3– exchanger AE2 playsan important role in the adaptation of bicarbonate absorptionin the TAL during chronic acid-base disturbances and high sodiumintake. The present study also emphasizes the contribution ofcortical TAL adaptation in the renal regulation of acid-basestatus.
The kidney controls systemic acid-base balance through the coordinatedregulation of multiple transport processes along the lengthof the nephron that together allow adaptation of HCO3–reabsorption [for review, see ref (1)]. Eighty-five percentof filtered bicarbonate is reabsorbed by the proximal tubule(PT), and the thick ascending limbs reabsorb the remaining 15%.In response to metabolic acidosis, HCO3– reabsorptionby PT cells is stimulated by an increase in expression and activityof the apical Na+/H+ exchanger NHE3, in conjunction with increasedactivity of the basolateral Na+-(HCO3–)n co-transporterkNBCe1 (2–4). Conversely, chronic metabolic alkalosisleads to the opposite regulation (5,6). Although bicarbonateabsorption occurs along the entire course of the thick ascendinglimb (TAL), its regulation has been studied most extensivelyin the medullary part of the TAL (MTAL). As in PT, chronic metabolicacidosis is associated with adaptive increases in MTAL bicarbonatereabsorption and enhanced apical NHE3 activity and expression(2,7,8). The MTAL response to chronic metabolic alkalosis dependson the experimental model. In rats with chronic chloride depletionmetabolic alkalosis with unaltered sodium intake, the HCO3–absorptive capacity of the MTAL is reduced (9). However, inrats with chronic metabolic alkalosis induced by chronic ingestionof NaHCO3, the HCO3– absorptive capacity of the MTAL isparadoxically enhanced (7), despite appropriate downregulationof apical NHE3 (10). The increased HCO3– absorption thatfollows NaHCO3 loading has been hypothesized to reflect adaptationof the MTAL to high sodium intake, an important determinantof MTAL bicarbonate absorption (7).
We have previously shown that the major basolateral HCO3–exit pathway of MTAL cells is via Cl–/HCO3– exchangeactivity most plausibly attributed to AE2 (11–13). AE2is also expressed in the cortical portion of the TAL (11). Itthus is possible that regulation of AE2 expression is involvedin adaptive change in TAL HCO3– reabsorption during acid-basedisorders and alterations in sodium intake. Therefore, the presentstudy compared changes in renal AE2 polypeptide abundance inresponse to chronic oral loading with NH4Cl, NaHCO3, KHCO3,NaCl, and KCl.
Animal Treatments
Male Sprague-Dawley rats (IFFA-CREDO, L’Arbresle, France)that weighed 240 to 260 g were used for this study.
First Protocol (NH4Cl, NaHCO3 and NaCl Loading).
Five independent series were performed. In each series, a controlgroup and three treated groups were handled in parallel overa 6-d period. Each group consisted of two rats. Treated ratswere given 0.28 M NH4Cl, NaHCO3, or NaCl and were fed ad libitumwith standard laboratory rat chow (Dietex, Saint-Gratien, France).Control rats were fed ad libitum with standard laboratory ratchow and had free access to distilled water for the same periodof time.
Second Protocol (KCl and KHCO3 Loading).
Two independent series were performed. In each series, a controlgroup and two treated groups were handled in parallel over a6-d period. Each group consisted of three rats. Treated ratswere given 0.28 M of either KCl or KHCO3 and were fed ad libitumwith standard laboratory rat chow (Dietex). Control rats werefed ad libitum with standard laboratory rat chow and had freeaccess to distilled water for the same period of time.
At the end of the experimental period, blood was collected byaortic puncture under anesthesia, provided by peritoneal injectionof pentobarbital sodium. The bladder was punctured just afteranesthesia, and urine was withdrawn. Kidneys were rapidly removedand immersed into ice-cold Hank’s modified solution thatcontained (in mM) 137 NaCl, 5.4 KCl, 25 NaHCO3, 0.3 Na2HPO4,0.4 KH2PO4, 0.5 MgCl2, 10 HEPES, 5 glucose, 1 leucine, and 1mg/ml BSA. Arterial pH, pCO2, and pO2 were measured with anAVL Compact 1 pH/blood-gas analyser (AVL Instruments Médicaux,Eragny-sur-Oise, France). Serum and urine electrolytes and creatininewere measured by standard methods with a Beckman LX20 autoanalyser(Coulter-Beckman, Villepinte, France) and Olympus AU 400 (Olympus,Rungis, France). Plasma aldosterone was measured by RIA (DPCDade Behring, La Défense, France). Urinary ammonia wasmeasured by the method of Berthelot (14). Urinary pH and HCO3–were measured with an AVL 555 Radiometer analyser (AVL InstrumentsMédicaux).
Preparation of Membrane Fractions
Excised kidneys were removed and cut into 5-mm slices. The cortexand the outer medulla (inner stripe) from the kidneys of tworats were excised under a stereoscopic microscope and placedinto ice-cold isolation buffer (250 mM sucrose and 20 mM Tris-HEPES[pH 7.4]) that contained protease inhibitors (in µg/ml):4 aprotinin, 4 leupeptin, 1.5 pepstatin A, and 28 4-(2-aminoethyl)-benzenesulfonylfluoride. Minced tissues were homogenized in a Dounce homogenizer(pestle A x 5) followed by five passes through a Teflon-glasshomogenizer rotating at 1000 rpm. The homogenate was centrifugedat 1000 x g for 10 min, and the supernatant was centrifugedat 100,000 x g for 20 min at 4°C. The pellet was resuspendedin isolation buffer. Protein concentration was determined usingthe Bradford protein assay.
Antibodies
Rabbit polyclonal antibody to mouse C-terminal AE2 amino acids1224 to 1237 has been previously characterized (11,13). Affinity-purifiedrabbit polyclonal antibodies that recognize the rat and mouseAE2a and AE2b isoforms (anti-AE2a/b [aa 101 to 117]) as describedpreviously by Frische et al. (15) were used for immunohistochemistryof rat kidney sections.
Immunoblot Analyses
Membrane proteins were solubilized in SDS-loading buffer (10mM Tris HCl [pH 6.8], 1% SDS, 2% dithiothreitol, 13% glycerol,and bromophenol blue), incubated at 20°C for 30 min. Electrophoresiswas initially performed for all samples on 7.5% polyacrylamideminigels (XCell SureLock Mini-cell; Invitrogen Life Technologies),which were stained with Coomassie blue to provide quantitativeassessment of loading. Figure 1A shows one representative experimentof Coomassie blue–stained control gels. Densitometry analyses(Figure 1B) revealed no variation in the Coomassie blue banddensities, indicating operationally identical protein loadingof the different samples. For immunoblotting, proteins weretransferred electrophoretically (XCell II Blot Module; InvitrogenLife Technologies) for 1.5 h at 4°C from unstained gelsto nitrocellulose membranes (Amersham, Arlington Heights, IL)and then stained with 0.5% Ponceau S in acetic acid to checkuniformity of protein transfer onto the nitrocellulose membrane.Posttransfer staining of gels with Coomassie blue confirmedcomplete protein transfer. Initial electrophoretic transfersused two membranes in tandem. The absence of transferred proteinon the membrane placed farther away from the gel indicated thatsignificant amounts of protein did not pass through the membraneto be probed. Membranes were first incubated in 5% nonfat drymilk in PBS (pH 7.4) for 1 h at room temperature to block nonspecificbinding of antibody, followed by overnight at 4°C with theanti-AE2 (aa 1224 to 1237) antibody (diluted 1:300 for the corticalsamples or 1:8000 for the medullary samples) in PBS that contained1% nonfat dry milk. After four 5-min washes in PBS that contained0.1% Tween-20, membranes were incubated with 1:3000 dilutionof goat anti-rabbit IgG conjugated to horseradish peroxidase(Bio-Rad, Hercules, CA) in PBS that contained 5% nonfat drymilk for 2 h at room temperature. Blots were washed as above,and luminol-enhanced chemiluminescence (NEN Life Science Products,Inc., Boston, MA) was used to visualize bound antibodies onHyperfilm ECL (Amersham). The autoradiographs were digitizedby laser scanner (Epson Perfection 1650; Epson), and bands werequantified using NIH Image software. Densitometric values werenormalized to the mean for the control group that was definedas 100%, and results were expressed as mean ± SEM.
Figure 1. Representative Coomassie blue–stained polyacrylamide gels used to control protein loading. The gels presented here show aliquots of the same samples presented in Figure 2. (A) Control rats (c) and acidotic rats (a). (B) Densitometric analyses of total electrophoresed protein separated on these gels established that the accompanying immunoblots (loaded identically; Figure 2A) were indeed uniformly loaded. Bars represent means ± SEM.
Figure 2. Effects of chronic metabolic acidosis induced by NH4Cl loading on AE2 protein abundance in the rat renal kidney. (A) Immunoblots of membrane fractions from the cortex and outer medulla (inner stripe) obtained from five acidotic rats (ac) and their respective control rats (c). Each lane was loaded with 10 µg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from five independent experiments revealed a significant increase in AE2 abundance in acidotic rats compared with control rats in the cortex and the outer medulla. Bars represent means ± SEM. *Statistically significant increase versus control (P < 0.05).
Immunohistochemistry of Rat Kidney Sections
Kidneys from three NH4Cl-loaded rats, six NaCl-loaded rats,and nine control rats were fixed with 4% paraformaldehyde. Kidneyslices that contained all kidney zones were dehydrated and embeddedin paraffin. The paraffin-embedded tissues were cut at 2-µmthickness on a rotary microtome (Leica, Wetzlar, Germany), dewaxedin xylene, and rehydrated through 99% ethanol to 96% ethanol.At this hydration level, endogenous peroxidase activity wasblocked by 30-min incubation in 0.3% H2O2 in methanol. Rehydrationwas completed through 96 and 70% ethanol. For revealing antigens,sections were placed in 10 mM Tris buffer (pH 9.0) that contained0.5 mM EGTA (Titriplex VI, Merck) and heated in a microwaveoven for 10 min. Nonspecific binding of Ig was prevented byincubating the sections in 50 mM NH4Cl for 30 min followed byblocking in PBS supplemented with 1% BSA, 0.05% saponin, and0.2% gelatin. Sections were incubated overnight at 4°C withaffinity-purified anti-AE2a/b (aa 101 to 117) antibodies diluted1:4000 (for the NaCl and NaCl controls) or 1:8000 (for NH4Cland NH4Cl controls) in 10 mM PBS (pH 7.4) that contained 0.1%Triton X-100 and 0.1% BSA, and for 2 h at room temperature withhorseradish peroxidase–linked goat anti-rabbit secondaryantibodies (P448; DAKO, Glostrup, Denmark). Labeling was visualizedby 3,3-diaminobenzidine technique, and the sections were counterstainedusing Mayer’s hematoxylin.
Statistical Analyses
All data are presented as means ± SEM. Comparisons amonggroups were assessed by ANOVA with Bonferroni correction orunpaired t test. P < 0.05 was considered significant.
AE2 Abundance Was Markedly Stimulated during Metabolic Acidosis in the Cortex and the Outer Medulla, Whereas Metabolic Alkalosis Induced by NaHCO3 Loading Increased AE2 Abundance Only in the Outer Medulla
To test whether chronic metabolic acidosis and alkalosis wouldchange the abundance of AE2 protein in the rat kidney, we preparedsemiquantitative immunoblots with membrane fractions from therenal cortex and outer medulla (inner stripe) of control ratsand of rats that were subjected to chronic loading with NH4Clor NaHCO3. Plasma electrolytes and acid-base status of the controland the NH4Cl- or NaHCO3-loaded groups are summarized in Table 1.Rats that were subjected to NH4Cl loading (5 to 9 mmol/d)developed metabolic acidosis. This was reflected in a significantdecrease in blood HCO3– concentration from the controlvalue of 27.7 ± 0.5 to 23.1 ± 1.3 mmol/L (P <0.05) and in a fall of blood pH from the control value of 7.37± 0.01 to 7.27 ± 0.03 (P < 0.05). Conversely,rats that were subjected to NaHCO3 loading developed metabolicalkalosis with a significant increase in plasma HCO3–concentration to 31.2 ± 0.8 mmol/L (P < 0.05, versuscontrol group values). As expected, acidotic rats exhibitedhigher urinary ammonium excretion than control rats (73 ±10 versus 9 ± 2 mmol/mmol creatinine; P < 0.05), whereasurinary ammonium excretion in alkalotic rats decreased to 1± 0.5 mmol/mmol creatinine (P < 0.05).
Table 1. Functional data in rats after chronic NaCl, NaHCO3, and NH4Cl loadinga
Figure 2A shows the results of immunoblots that were probedwith anti-AE2 antibody (aa 1224 to 1237). As shown in Figure 2and Table 2, metabolic acidosis induced a large increase inAE2 abundance in the cortex (644 ± 37% in the acidosisgroup versus 100 ± 15% in the control group; P < 0.001)and the outer medulla (238 ± 45% in the acidosis groupversus 100 ± 15% in the control group; P < 0.05).Semiquantitative immunoblotting of membrane fractions from thealkalotic rats showed that AE2 abundance was significantly increasedin the outer medulla (176 ± 25% in the alkalosis groupversus 100 ± 15% in the control group; P < 0.05) butnot in the cortex (126 ± 21% versus 100 ± 10%in the control group; NS; Figure 3, Table 2).
Figure 3. Effects of chronic metabolic alkalosis induced by NaHCO3 loading on AE2 protein abundance in the rat kidney. (A) Immunoblots of membrane fractions from the cortex and outer medulla (inner stripe) obtained from five alkalotic rats (al) and their respective controls (c). Each lane was loaded with 10 µg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from five independent experiments showing that the abundance of AE2 in alkalotic rats was significantly increased in the outer medulla but not in the cortex when compared with control rats. Bars represent means ± SEM. *Statistically significant increase versus control (P < 0.05).
AE2 Abundance Was Also Increased in Response to High NaCl Intake
As the effect of NaHCO3 loading on AE2 abundance could havebeen the result of the increase in sodium intake rather thanthe effect of increased bicarbonatemia, we also tested in parallelthe effect on AE2 abundance of the same Na+ intake administeredas NaCl loading. Table 1 shows functional data obtained in ratsthat were subjected to chronic NaCl loading. As expected, urinarysodium excretion was markedly increased in rats that were exposedto high sodium intake (NaCl or NaHCO3 loading) compared withcontrol rats. Importantly, there was no difference between sodiumexcretion of NaCl-loaded rats compared with NaHCO3-loaded rats.In contrast to NaHCO3 loading, NaCl loading did not change bloodpH, plasma HCO3–, and serum chloride concentrations whencompared with control rats.
Figure 4 and Table 2 show that chronic NaCl loading markedlystimulated AE2 abundance in the outer medulla (202 ±11% in NaCl-loaded rats versus 100 ± 13% in the controlgroup; P < 0.001). In contrast to NaHCO3 loading, NaCl loadingalso increased AE2 abundance to the same extent in the cortex(201 ± 26% in NaCl-loaded rats versus 100 ± 7%in the control group; P < 0.01). Taken together, the resultsshown in Figures 3 and 4 suggest that increased Na+ intake itselfincreases kidney AE2 abundance.
Figure 4. Effects of chronic sodium loading induced by NaCl loading on AE2 protein abundance in the rat kidney. (A) Immunoblots of membrane fractions from the cortex and the outer medulla (inner stripe) obtained from a control rat (c) and its pair-fed NaCl-loaded rat (na). Each lane was loaded with 10 µg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from five independent experiments showing that the abundance of AE2 in alkalotic rats was significantly increased in the cortex and the outer medulla compared with control rats. Bars represent means ± SEM. *Statistically significant increase versus control (P < 0.05).
AE2 in the Renal Cortex Was Downregulated in Response to Chronic KHCO3 Loading
Because Na+ loadings experiments, administered with either Cl–or HCO3–, indicated that Na+ may exert an independentstimulatory effect on AE2, we hypothesized that the expecteddownregulation of AE2 in response to an increased bicarbonatemiamay have been masked by an opposing upregulation as a resultof the parallel increase in Na+ intake during the NaHCO3-loadingprotocol. To test this hypothesis, we next studied the effectsof the same HCO3– load administered with K+ as the accompanyingcation rather than Na+. Functional data are presented in Table 3.Rats that were subjected to KHCO3 loading developed metabolicalkalosis with an increase in plasma HCO3– concentrationfrom the control value of 24.7 ± 0.9 to 29.3 ±0.9 mmol/L in treated rats (P < 0.05) and exhibited a significantlyhigher urinary pH and bicarbonate excretion. Figure 5 and Table 2show that AE2 protein abundance in the renal cortex was markedlydecreased in response to chronic KHCO3 loading (49 ±8% in KHCO3-loaded rats versus 100 ± 7% in control group;P < 0,01). No significant change was observed in the abundanceof AE2 in the outer medulla after KHCO3 loading (130 ±19% in KHCO3-loaded rats versus 100 ± 10% in controlgroup).
Figure 5. Effects of chronic KHCO3 loading on AE2 protein abundance in the rat kidney. (A) Immunoblots of membrane fractions from the cortex obtained from six KHCO3-loaded rats (k) and their respective controls (c). Each lane was loaded with 10 µg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from six independent experiments showing that the abundance of AE2 in KHCO3-loaded rats was significantly decreased in the cortex but not in the outer medulla when compared with control rats. Bars represent means ± SEM. *Statistically significant increase versus control (P < 0.05).
Rats with Chronic KCl Loading Did not Experience Changes in the Abundance of AE2
Our findings in the renal cortex could also be interpreted toindicate that Cl– loading upregulates AE2 independentof acid-base or Na+ balance. Indeed, as summarized in Table 2,we observed upregulation of AE2 expression in response toNH4Cl and NaCl loading, whereas there was no effect on AE2 expressionwhen sodium loading was achieved with NaHCO3. To test the hypothesisthat AE2 expression is regulated by chloride balance, we nextstudied the effects of an equivalent chloride load administeredwith K+ as the accompanying cation rather than Na+. As expected,high dietary KCl intake markedly increased urinary chlorideexcretion from the control value of 10 ± 2 to 60 ±9 mmol/L (P < 0.05; Table 3), an increase similar in magnitudeto those observed during loading with NaCl and with NH4Cl (seeTable 1). Importantly, the potassium load reflected by urinarypotassium excretion was identical to that achieved by KHCO3loading, but the KCl treatment did not alter acid-base status(Table 3). As shown in Figure 6, KCl loading had no significanteffect on AE2 abundance in either the renal cortex (129 ±10% in KCl-loaded rats versus 100 ± 9% in control group;NS) or the outer medulla (143 ± 18% in KCl-loaded ratsversus 100 ± 15% in control group; NS). The data thusindicate that neither Cl– nor K+ loads are important regulatorsof AE2 abundance.
Figure 6. Effects of chronic KCl loading on AE2 protein abundance in the rat kidney. (A) Immunoblots of membrane fractions from the cortex obtained from six KCl-loaded rats (kcl) and their respective controls (c). Each lane was loaded with 10 µg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from six independent experiments showing that the abundance of AE2 in KCl-loaded rats is not modified in the cortex or in the outer medulla as compared with control rats. Bars represent means ± SEM.
Taken together, our results show that AE2 protein expressionis appropriately regulated by acid-base status in the renalcortex, i.e., stimulated by metabolic acidosis and inhibitedby alkalemia. In the outer medulla, the effect of acid-basebalance on AE2 abundance is limited to the stimulatory effectof acidosis. Nevertheless, our experiments also demonstratethat acid-base balance is not the only determinant of AE2 regulation.Indeed, Na+ intake seems to exert a potent effect also on AE2.
Immunohistochemistry
AE2 is mainly expressed on the basolateral membranes of theTAL, macula densa, and inner medullary collecting duct. To determineat which of these sites NH4Cl and NaCl loading increased AE2expression, we performed immunohistochemistry in control andtreated rats. As previously shown (11,15), AE2 immunolabelingwith the AE2a/b antibody in kidneys from control rats was evidentin basolateral domains of both cortical and medullary segmentsof TAL, as well as in the macula densa, and also in the terminalcollecting ducts. After the different treatments, all of thedetectable changes in the intensity of AE2 labeling were observedin the TAL; we also did not observe the apparition of an AE2labeling in cells that did not express AE2 in the basal state.Kidneys from NaCl-loaded rats showed markedly increased intensityof AE2 labeling in basolateral domains of MTAL, with a lesserincrease noted in cortical TAL (CTAL; Figure 7). In sectionsfrom NH4Cl-loaded rats, a marked increase in labeling intensitywas evident in the CTAL, and the labeling in CTAL extended tomore distal parts of the CTAL (Figure 8B) as compared with controls(Figure 8A). No changes in AE2 labeling were observed in MTALin NH4Cl-loaded rats (Figure 8D) compared with controls (Figure 8C).
Figure 7. AE2 immunolabeling in kidneys from control (A and C) and NaCl-loaded (B and D) rats. In kidneys from NaCl-loaded rats, the intensity of AE2 labeling in basolateral domains of cortical thick ascending limb (CTAL; A and B) was weakly increased, whereas a marked increase in labeling intensity was evident in medullary thick ascending limb (MTAL; C and D). CD, collecting duct. Scale bars = 50 µm.
Figure 8. AE2 immunolabeling in kidneys from control rats (A and C) and NH4Cl-loaded rats (B and D) rats. In cortex, AE2 labeling was increased in the CTAL in NH4Cl-loaded rats (B) in comparison with controls (A). In MTAL, no changes were seen in the intensity of AE2 labeling in NH4Cl-loaded rats (D) when compared with controls (C). Scale bars = 50 µm.
The AE2 Cl–/HCO3– exchanger polypeptides are expressedthroughout the distal nephron in epithelial cell basolateralmembranes, with highest expression in the MTAL, macula densa,and inner medullary collecting duct (11,15). Lower levels ofAE2 expression were detected in CTAL, thin limbs, and principalcells of the outer medullary collecting duct (11). AE2 may contributeto transepithelial bicarbonate transport, to chloride secretion,or to "housekeeping functions" such as cellular regulatory volumeincrease and pHi regulation. In two studies using basolateralmembrane vesicles isolated from a purified MTAL tubule suspension,we previously demonstrated that the main basolateral HCO3–efflux pathway in MTAL cells is via Cl–/HCO3– exchangeactivity attributable to AE2 (12,13). An additional study fromour laboratory confirmed in isolated, intact, microperfusedMTAL that the main basolateral bicarbonate efflux pathway wasCl– dependent, Na+ independent, and 4,4'-diisothiocyanostilbene-2,2'-disulfonicacid (DIDS) sensitive, properties shared with AE2 (16).
The present study examined the regulation of AE2 expressionin rats that were subjected to chronic acid-base perturbationsand increased sodium intake. Chronic metabolic acidosis markedlyincreased AE2 abundance in the renal cortex and outer medulla,as detected by immunoblot. Chronic sodium loading, as eitherNaHCO3 or NaCl, increased AE2 abundance to a similar extentin the outer medulla, whereas in the cortex, only chronic NaClloading led to an increase in AE2 protein expression. Immunohistochemistrywas performed to define the nephron segments in which AE2 abundancewas changed. First, the immunoblot data can be ascribed to changesin the basolateral membrane of the TAL because AE2 was not foundto be upregulated in other segments. Second, immunoblot andimmunohistochemistry findings were in agreement, with the exceptionof those of the outer medulla after NH4Cl loading. Discrepancybetween immunoblotting and immunohistochemistry regarding AE2regulation in the inner stripe of the outer medulla could beexplained by the lower sensitivity of immunohistochemistry thanof immunoblotting for quantifying differences in protein abundance.However, although AE2 immunostaining intensity in TAL of theinner stripe of the outer medulla was not increased in NH4Cl-loadedrats, the tubules seemed larger than in control rats (i.e.,hypertrophic). This latter observation could explain detectionof increased AE2 abundance only by immunoblot. In contrast,in NaCl-loaded rats, TAL show a marked increase in immunostainingintensity, confirming that AE2 protein abundance was specificallyupregulated in this condition in the outer medulla. Finally,the immunoblot data suggest considerable variation among individualsin the magnitude of AE2 upregulation in the outer medulla (seeFigure 2). This could explain why individual fixed kidneys studiedby immunohistochemistry can show little or no regulation.
In a recent study, Frische et al. (15) determined the renalexpression pattern of polypeptide products of the AE2 transcriptvariants AE2a and AE2b and the regulation of AE2 polypeptidesby chronic acid loading. In that study, levels of AE2 polypeptidewere markedly increased in CTAL in response to chronic administrationof NH4Cl or HCl. In contrast to our current results, Frischeet al. observed no change in AE2 protein level in the outermedulla. However, whereas the 6-d acid-loaded rats in the seriesstudied by Frische et al. exhibited only a decrease in urinarypH without altered plasma acid-base status [see the Methodssection in reference (15)], our "acidotic" rats exhibited afrank metabolic acidosis, with significant decreases in bothplasma pH and bicarbonate concentration (see Table 1). The greaterdegree of acidosis experienced by the animals in our currentstudy may account for the greater increase in AE2 expression[in the renal cortex: 644 versus 203% in ref (15)], includingupregulation of AE2 not only in CTAL but also in MTAL. Anotherpossible explanation for the detection of increased AE2 in MTALin our current study may lie in our use of immunoblot experimentsof a particular fraction more highly enriched in plasma membrane.Indeed, comparison of whole homogenates from the inner stripeof outer medulla of our control and NH4Cl-loaded rats revealedonly minimal increase in AE2 abundance (132 ± 7% in theacidotic group versus 100 ± 14% in the control group;NS), similar to the findings of Frische et al. (15).
The upregulation of AE2 seen in the MTAL in response to loadingwith NH4Cl and to Na+ loading is a novel finding. Good (7) previouslydemonstrated in isolated MTAL microperfused in vitro that bicarbonateabsorption is increased by chronic NH4Cl loading. He also observedthat chronic sodium intake increased bicarbonate absorptivecapacity to the same extent when sodium was administered aseither sodium bicarbonate or sodium chloride. Thus, the increasein AE2 abundance demonstrated here is likely to have contributedto the previously observed increase in HCO3– absorption.Upregulation of basolateral bicarbonate efflux through AE2-mediatedCl–/HCO3– exchange may, with coordinated stimulationof apical NHE3-mediated Na+/H+ exchange, contribute to enhancedtransepithelial bicarbonate reabsorption during metabolic acidosis(10). In contrast, the stimulatory effect of chronic sodiumloading on bicarbonate reabsorption could not be due to thesame mechanism because NHE3 abundance in TAL is decreased orunaltered in the TAL during chronic NaHCO3 or NaCl loading (10,17).During chronic changes in sodium bicarbonate loading, the increasesin bicarbonate absorption and in AE2 abundance seem inappropriatefor maintaining acid-base balance and likely reflect MTAL adaptationto high sodium intake. When chloride or HCO3– loads wereadministered as K+ salts, no change in AE2 abundance was seenin the outer medulla, demonstrating the importance of increasedsodium intake in AE2 adaptation. These results also show thatthe high chloride load that accompanies NH4+ has no effect onAE2 expression and so demonstrate that acidosis per se regulatesAE2 expression. Although chronic KHCO3 loading and its mildmetabolic alkalosis did not lead to AE2 downregulation in theouter medulla, severe alkalemia was shown to decrease bicarbonateabsorptive capacity in the MTAL (9). It thus seems that acid-basestatus is another important determinant for AE2 adaptation.
In the present study, NaHCO3 loading was associated with a suggestivebut nonsignificant trend toward hypokalemia (Table 1), reflectinga possible potassium depletion. Hypokalemia is known to stimulateHCO3– reabsorption (18,19). NHE3, which plays an importantrole in the process of HCO3– reabsorption, is upregulatedafter 4 d of dietary K+ depletion–induced hypokalemia(20). Thus, upregulation of AE2 abundance in NaHCO3-loaded ratsmay also be caused by or reflect an effect of K+ depletion andits associated increased HCO3– absorption. Taken together,these results support the hypothesis that HCO3 transport byMTAL cells through AE2 regulation is specifically regulatedby changes in sodium intake and acid-base status and not bychanges in chloride and potassium intake.
The CTAL response to salt loading differed from that of theMTAL. NaHCO3 loading produced no change in CTAL AE2 abundance.In contrast, the effect of NH4Cl loading was much greater inCTAL than in MTAL. The unchanged renal cortical AE2 abundanceafter NaHCO3 loading likely reflects the sum of two processes:AE2 downregulation by alkali load and AE2 upregulation by theconcomitant Na load. This possibility is further supported bythe decrease in renal cortical AE2 abundance in response toKHCO3 loading and by the absence of effect of KCl loading. Thus,AE2 expression in the renal cortex may be particularly sensitiveto changes in acid-base status.
Because metabolic acidosis decreases filtered bicarbonate loadwhile increasing proximal bicarbonate reabsorptive capacity,upregulation of TAL bicarbonate transport in metabolic acidosismight seem unnecessary, but the increase in bicarbonate transportcapacity during acidosis could enhance urinary acid excretionby maintaining a low luminal bicarbonate concentration alongthe TAL and further limit bicarbonate delivery to the distaltubule. In particular, the adaptive increase in bicarbonateabsorption (e.g., AE2 and NHE3 proteins adaptation) may be coupledto ammonium handling. As previously proposed by Good (7), TALbicarbonate absorptive capacity may compensate for the acidosis-inducedincrease in ammonium absorption along the TAL that would tendto alkalinize the luminal fluid were not H+ secretion simultaneouslyincreased.
The present study provides additional support for the importanceof cortical distal nephron segments in the renal regulationof acid-base status. Previous studies have shown that chronicmetabolic acidosis markedly increases ammonia secretion alongthe collecting duct (21–24), but a microcatheterizationstudy suggested that in acidotic rats, the bulk of ammonia reentersthe luminal fluid primarily in the cortical collecting duct(22). In that study, the amount of ammonium secreted into thecortical collecting duct of acidotic rats was 15-fold greaterthan that of control rats, whereas the ammonium added to themedullary collecting duct was increased only three- to fourfold.Thus, if HCO3– reabsorption by the TAL is regulated tosupport parallel increase in ammonium transport, then a correspondinglymarked increase in HCO3– transport capacity should occurin the cortical portion of the TAL.
The dependence of both TAL luminal acidification and AE2 upregulationon sodium intake suggests that the TAL segment may play an importantrole in maintaining acid-base balance when sodium chloride intakeis altered. In the setting of elevated salt intake and extracellularfluid volume, an increase in bicarbonate absorption in TAL couldoppose a reduction in collecting duct proton secretion as aresult of decreased plasma aldosterone levels to maintain urinaryacid excretion. The mediators of the TAL adaptation in responseto changes in sodium intake have not been determined in thisstudy.
In summary, our results demonstrate that acid-base balance andsodium intake are important and independent determinants ofAE2 regulation in the TAL. This study also supports the importanceof the CTAL in the renal regulation of acid-base status. TheCTAL and MTAL may play an especially important role in maintainingacid-base balance when sodium intake is altered. We proposethat this process involves changes in the basolateral expressionof the Cl–/HCO3– exchanger AE.
Acknowledgments
The Water and Salt Research Center at the University of Aarhusis established and supported by the Danish National ResearchFoundation (Danmarks Grundforskningsfond). Additional fundingwas provided by the European Commission (QLRT-2000-00987).
We thank Susan M. Wall for providing the AE2a/b antibody andInger Merete Paulsen, Ida Maria Jalk, and Lotte Vallentin Holbechfor expert technical assistance.
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Received for publication December 17, 2003.
Accepted for publication September 10, 2004.
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Abstract
Introduction
suggest that increased Na+ intake itself increases kidney AE2 abundance. 
