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Adaptation to metabolic acidosis and its recovery are associated with changes in anion exchanger distribution and expression in the cortical collecting duct

2010; Elsevier BV; Volume: 78; Issue: 10 Linguagem: Inglês

10.1038/ki.2010.195

1523-1755

Jeffrey M. Purkerson, Shuichi Tsuruoka, D. Zachary Suter, Aya Nakamori, George J. Schwartz,

Pancreatic function and diabetes

It is well known that acid/base disturbances modulate proton/bicarbonate transport in the cortical collecting duct. To study the adaptation further we measured the effect of three days of acidosis followed by the rapid recovery from this acidosis on the number and type of intercalated cells in the rabbit cortical collecting duct. Immunofluorescence was used to determine the expression of apical pendrin in β-intercalated cells and the basolateral anion exchanger (AE1) in α-intercalated cells. Acidosis resulted in decreased bicarbonate and increased proton secretion, which correlated with reduced pendrin expression and the number of pendrin-positive cells, as well as decreased pendrin mRNA and protein abundance in this nephron segment. There was a concomitant increase of basolateral AE1 and α-cell number. Intercalated cell proliferation did not seem to play a role in the adaptation to acidosis. Alkali loading for 6–20 h after acidosis doubled the bicarbonate secretory flux and reduced proton secretion. Pendrin and AE1 expression patterns returned to control levels, demonstrating that adaptive changes by intercalated cells are rapidly reversible. Thus, regulation of intercalated cell anion exchanger expression and distribution plays a key role in adaptation of the cortical collecting duct to perturbations of acid/base. It is well known that acid/base disturbances modulate proton/bicarbonate transport in the cortical collecting duct. To study the adaptation further we measured the effect of three days of acidosis followed by the rapid recovery from this acidosis on the number and type of intercalated cells in the rabbit cortical collecting duct. Immunofluorescence was used to determine the expression of apical pendrin in β-intercalated cells and the basolateral anion exchanger (AE1) in α-intercalated cells. Acidosis resulted in decreased bicarbonate and increased proton secretion, which correlated with reduced pendrin expression and the number of pendrin-positive cells, as well as decreased pendrin mRNA and protein abundance in this nephron segment. There was a concomitant increase of basolateral AE1 and α-cell number. Intercalated cell proliferation did not seem to play a role in the adaptation to acidosis. Alkali loading for 6–20 h after acidosis doubled the bicarbonate secretory flux and reduced proton secretion. Pendrin and AE1 expression patterns returned to control levels, demonstrating that adaptive changes by intercalated cells are rapidly reversible. Thus, regulation of intercalated cell anion exchanger expression and distribution plays a key role in adaptation of the cortical collecting duct to perturbations of acid/base. The kidney controls acid/base balance, and the ultimate fine tuning of renal acid/base transport occurs in the cortical collecting duct (CCD). Intercalated cells, which make up a third of cells of the CCD,1.Schwartz G.J. Barasch J. Al-Awqati Q. Plasticity of functional epithelial polarity.Nature. 1985; 318: 368-371Crossref PubMed Scopus (243) Google Scholar are responsible for acid/base transport; α-intercalated cells secrete protons and β-intercalated cells secrete bicarbonate.2.Wagner C.A. Devuyst O. Bourgeois S. et al.Regulated acid-base transport in the collecting duct.Pflugers Arch. 2009; 458: 137-156Crossref PubMed Scopus (131) Google Scholar,3.Schwartz G.J. Tsuruoka S. Vijayakumar S. et al.Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.J Clin Invest. 2002; 109: 89-99Crossref PubMed Scopus (85) Google Scholar Type α-intercalated cells have apical H+-ATPases and basolateral anion exchangers (AE1), whereas β-intercalated cells express apical anion exchangers (pendrin) and basolateral H+-ATPases. We have examined changes in the physiology of the CCD during metabolic acidosis in vivo4.Satlin L.M. Schwartz G.J. Cellular remodeling of HCO3--secreting cells in rabbit renal collecting duct in response to an acidic environment.J Cell Biol. 1989; 109: 1279-1288Crossref PubMed Scopus (64) Google Scholar and in vitro.3.Schwartz G.J. Tsuruoka S. Vijayakumar S. et al.Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.J Clin Invest. 2002; 109: 89-99Crossref PubMed Scopus (85) Google Scholar,5.Tsuruoka S. Schwartz G.J. Adaptation of rabbit cortical collecting duct HCO3- transport to metabolic acidosis in vitro.J Clin Invest. 1996; 97: 1076-1084Crossref PubMed Scopus (47) Google Scholar Indeed, there is an increase in H+ secretion and a decrease in HCO3- secretion in CCDs taken from acidotic rabbits,4.Satlin L.M. Schwartz G.J. Cellular remodeling of HCO3--secreting cells in rabbit renal collecting duct in response to an acidic environment.J Cell Biol. 1989; 109: 1279-1288Crossref PubMed Scopus (64) Google Scholar or after acid incubation.3.Schwartz G.J. Tsuruoka S. Vijayakumar S. et al.Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.J Clin Invest. 2002; 109: 89-99Crossref PubMed Scopus (85) Google Scholar,5.Tsuruoka S. Schwartz G.J. Adaptation of rabbit cortical collecting duct HCO3- transport to metabolic acidosis in vitro.J Clin Invest. 1996; 97: 1076-1084Crossref PubMed Scopus (47) Google Scholar In addition, there is a loss of apical anion exchange in β-intercalated cells and a reduction of the apical membrane that binds peanut agglutinin.3.Schwartz G.J. Tsuruoka S. Vijayakumar S. et al.Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.J Clin Invest. 2002; 109: 89-99Crossref PubMed Scopus (85) Google Scholar Recent studies suggest that the extracellular matrix (ECM) protein hensin is deposited in the ECM underneath β-intercalated cells and facilitates their adaptation to metabolic acidosis.3.Schwartz G.J. Tsuruoka S. Vijayakumar S. et al.Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.J Clin Invest. 2002; 109: 89-99Crossref PubMed Scopus (85) Google Scholar In fact, a third of such adapting cells not only lost apical anion exchange but established basolateral anion exchange, suggesting a reversal in functional polarity. Such an acid-induced insertion/activation of basolateral anion exchangers has also been observed by Merot et al.6.Merot J. Giebisch G. Geibel J. Intracellular acidification induces Cl/HCO3 exchange activity in the basolateral membrane of ß-intercalated cells of the rabbit cortical collecting duct.J Membrane Biol. 1997; 159: 253-262Crossref PubMed Scopus (7) Google Scholar We surmised that the change in β-intercalated cell physiology was induced by the deposition of hensin in the ECM underlying these adapting cells. Anti-hensin antibodies,3.Schwartz G.J. Tsuruoka S. Vijayakumar S. et al.Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.J Clin Invest. 2002; 109: 89-99Crossref PubMed Scopus (85) Google Scholar as well as inhibitors of cyclophilin PPIase (peptidylprolyl cis/trans isomerase) activity that block hensin secretion,7.Peng H. Vijayakumar S. Schiene-Fischer C. et al.Secreted cyclophilin A, a peptidylprolyl cis-trans isomerase, mediates matrix assembly of hensin, a protein implicated in epithelial differentiation.J Biol Chem. 2009; 284: 6465-6475Crossref PubMed Scopus (35) Google Scholar inhibit acid-induced changes in intercalated cell physiology.8.Watanabe S. Tsuruoka S. Vijayakumar S. et al.Cyclosporin A produces distal renal tubular acidosis by blocking peptidyl prolyl cis-trans isomerase activity of cyclophilin.Am J Physiol. 2005; 288: F40-F47Crossref PubMed Scopus (50) Google Scholar Hensin is expressed in many epithelial cells and serves to induce a differentiated phenotype,9.Vijayakumar S. Takito J. Hikita C. et al.Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation.J Cell Biol. 1999; 144: 1057-1067Crossref PubMed Scopus (79) Google Scholar, 10.Hikita C. Vijayakumar S. Takito J. et al.Induction of terminal differentiation in epithelial cells requires polymerization of hensin by galectin 3.J Cell Biol. 2000; 151: 1235-1246Crossref PubMed Scopus (123) Google Scholar, 11.Takito J. Al-Awqati Q. Conversion of ES cells to columnar epithelia by hensin and to squamous epithelia by laminin.J Cell Biol. 2004; 166: 1093-1102Crossref PubMed Scopus (52) Google Scholar suggesting that differentiation might be difficult to reverse once the matrix is laid down. It would follow that reversal of the acidosis might be associated with a significant delay in adaptive changes of intercalated cells. Accordingly, we attempted to reverse acidosis by abruptly changing the diet of the rabbits, and examined changes in cell physiology, transport, and phenotype. Would such ‘terminally differentiated’ intercalated cells respond rapidly to the correction of acidosis? The purpose of this study was to characterize in rabbits the changes in pendrin and AE1 distribution, expression, and synthesis, and in bicarbonate transport, in response to acid loading in vivo and then to determine what occurs within 12–16 h of NaHCO3 administration, when acidosis has been rapidly reversed. In the rabbit CCD the adaptation to acidosis involves compensatory changes in H+/HCO3 transport by α- and β-intercalated cells, respectively, that is regulated in part by ETB receptor signaling12.Tsuruoka S. Watanabe S. Purkerson J.M. et al.Endothelin and nitric oxide mediate adaptation of the cortical collecting duct to metabolic acidosis.Am J Physiol Renal Physiol. 2006; 291: F866-F873Crossref PubMed Scopus (18) Google Scholar and changes in the composition of ECM hensin predominantly surrounding β-intercalated cells.3.Schwartz G.J. Tsuruoka S. Vijayakumar S. et al.Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.J Clin Invest. 2002; 109: 89-99Crossref PubMed Scopus (85) Google Scholar Because it has been suggested that ECM hensin and galectin-3 mediate signals that promote acquisition of a terminally differentiated epithelial cell phenotype,9.Vijayakumar S. Takito J. Hikita C. et al.Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation.J Cell Biol. 1999; 144: 1057-1067Crossref PubMed Scopus (79) Google Scholar,10.Hikita C. Vijayakumar S. Takito J. et al.Induction of terminal differentiation in epithelial cells requires polymerization of hensin by galectin 3.J Cell Biol. 2000; 151: 1235-1246Crossref PubMed Scopus (123) Google Scholar it would seem that once hensin is deposited in the ECM, a cell could not rapidly de-differentiate. Accordingly, we sought to develop an in vivo model in which we could identify the parameters associated with reversible adaptive changes in intercalated cell phenotypes that define the response of α- and β-intercalated cells to perturbations in acid/base status. In this study we have compared the intercalated cell phenotypes in the CCD of normal rabbits with those from rabbits administered NH4Cl for 3 days (acidosis) versus rabbits administered NH4Cl for 3 days and abruptly transitioned to NaHCO3 for 12–18 h (recovery). As shown in Figure 1, normal rabbits fed an alkaline ash diet showed serum bicarbonate levels between 25 and 30 mmol/l (lower panel) with an alkaline urine (pH 8.1±0.1, upper panel), whereas NH4Cl loading induced marked acidosis characterized by reduction of serum bicarbonate to 15–16 mmol/l (lower panel) and acidification of urine to pH below 5 (upper panel). In rabbits transitioned from NH4Cl to NaHCO3 (recovery), the serum bicarbonate returned to essentially ‘normal’ levels (normal vs recovery; P-value=0.092) and the urine pH values recovered to 7.8+0.1 (normal vs recovery; P-value=0.0190). As acidosis reduces bicarbonate secretion in the rabbit,3.Schwartz G.J. Tsuruoka S. Vijayakumar S. et al.Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.J Clin Invest. 2002; 109: 89-99Crossref PubMed Scopus (85) Google Scholar, 4.Satlin L.M. Schwartz G.J. Cellular remodeling of HCO3--secreting cells in rabbit renal collecting duct in response to an acidic environment.J Cell Biol. 1989; 109: 1279-1288Crossref PubMed Scopus (64) Google Scholar, 5.Tsuruoka S. Schwartz G.J. Adaptation of rabbit cortical collecting duct HCO3- transport to metabolic acidosis in vitro.J Clin Invest. 1996; 97: 1076-1084Crossref PubMed Scopus (47) Google Scholar these data suggest that a significant amount of bicarbonate secretion had been rapidly restored in the CCD within 12–18 h after bicarbonate administration. Rabbit weights measured just before killing were not significantly different among acid/base conditions: normal=2.53±0.08, acidotic=2.53±0.08, and recovery=2.33±0.05 (normal vs recovery, acidosis vs recovery; P>0.04). These data suggest that dehydration was not an important factor in the generation of acidosis.13.Amlal H. Sheriff S. Soleimani M. Upregulation of collecting duct aquaporin-2 by metabolic acidosis: role of vasopressin.Am J Physiol Cell Physiol. 2004; 286: C1019-C1030Crossref PubMed Scopus (38) Google Scholar Pendrin (SLC26A4) has been identified as the apical chloride/bicarbonate exchanger expressed by β-intercalated cells,14.Royaux I.E. Wall S.M. Karniski L.P. et al.Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion.Proc Natl Acad Sci USA. 2001; 98: 4221-4226Crossref PubMed Scopus (446) Google Scholar,15.Soleimani M. Greeley T. Petrovic S. et al.Pendrin: an apical Cl-/OH-/HCO3- exchanger in the kidney cortex.Am J Physiol. 2001; 280: F356-F364PubMed Google Scholar and both pendrin expression and subcellular localization are regulated by changes in acid/base status.16.Petrovic S. Wang Z. Ma L. et al.Regulation of the apical Cl-/HCO3- exchanger pendrin in rat cortical collecting duct in metabolic acidosis.Am J Physiol. 2003; 284: F103-F112Crossref PubMed Scopus (42) Google Scholar, 17.Wagner C.A. Finberg K.E. Stehberger P.A. et al.Regulation of the expression of the Cl-/anion exchanger pendrin in mouse kidney by acid-base status.Kidney Int. 2002; 62: 2109-2117Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 18.Frische S. Kwon T.H. Frokiaer J. et al.Regulated expression of pendrin in rat kidney in response to chronic NH4Cl or NaHCO3 loading.Am J Physiol. 2003; 284: F584-F593Crossref PubMed Scopus (102) Google Scholar In the rabbit CCD, pendrin expression is observed as a brightly stained crescent shape (pendrin cap) on the apical surface of β-intercalated cells in the outer cortical region of kidney sections stained with anti-pendrin antibody (Figure 2a). In this study we compared pendrin expression in the CCDs of the outer cortex by immunostaining of kidney sections prepared from normal, acid-loaded (acidosis) rabbits, and acid followed by alkali-loaded (recovery) rabbits. Tables 1 and 2 list the numbers of rabbits and kidney sections as well as the total number CCDs and/or cells that comprise the results summarized in Figures 2 and 3. Acidosis reduced both the apparent level of pendrin expression and the size of pendrin caps. Pendrin cap staining intensity was reduced in half (Figure 2b), and pendrin cap size distribution was shifted to the left (reduced, Figure 2c), which most likely reflects apical endocytosis of the transporter. In addition, acidosis, whether extended over a 3- or 7-day period, reduced the apparent number of pendrin-positive cells in CCDs of the outer cortex by ∼1 cell per 100 μm length of CCD (Figure 2d).Table 1Summary of immunofluorescent staining analysisParameterAcid/base statusRabbit N=Sections/rabbitSections, total no.Image no./magn.No. of CCDsNo. of cellsPendrin cap intensity/sizeNormal32–3726/ × 400—397Acidosis41–3727/ × 400—462Recovery32–3726/ × 400—526AE1 basolateral intensityNormal32621/ × 400—186Acidosis42828/ × 400—293Recovery32624/ × 400—182AE1 distributionNormal32–5933/ × 400—194Acidosis42–51140/ × 400—307Recovery32–5936/ × 400—230Mitotic figures (Sytox)Acidosis61–28273/ × 1002487—Ki-67Acidosis224—232—Abbreviations: AE1, anion exchanger; CCD, cortical collecting duct; magn., magnification. Open table in a new tab Table 2Summary of CD cell enumerationParameterRegionAcid/base statusRabbit N=Sections, total no.Image no.No. of CDsNo. of cellsPendrin+ cells per 100 μmCortexNormal34231684535Acidosis342717844147-day Acid.24171313290Recovery34302135646AE1+ cells per 100 μmCortexNormal34301871441Acidosis342815512957-day Acid.24523735400Recovery34261891611AE1+ cells per 100 μmMedullaNormal34181201556Acidosis341512020807-day Acid.24211523004Recovery341469913AQP2+ cells per 100 μmCortexNormal34341506478Acidosis34239751997-day Acid.24281387787Recovery3426883663AQP2+ cells per 100 μmMedullaNormal34231728538Acidosis341715910,1617-day Acid.2417139139Recovery3420119119Abbreviations: Acid., acidosis; AE1, anion exchanger; AQP2, aquaporin 2; CD, collecting duct. Open table in a new tab Figure 3Changes in acid/base status induces redistribution of anion exchanger (AE1) expression and changes in the apparent number of α-intercalated cells. (a) Representative digital images of AE1 staining (green) within the outer cortex of kidney sections prepared from rabbits with indicated acid/base status. Arrows point to cells positively stained for AE1, whereas the white lines delineate a 200-μm calibration mark stamped onto the digital image. (b) Recovery from acidosis reverses the increase in basolateral AE1 expression induced by acidosis. Fluorescent intensity of basolateral AE1 staining was measured with ImageJ using kidney sections from at least three rabbits per condition (Table 1). Basolateral AE1 staining intensity in sections from normals was set to 1, and the mean±s.e. intensities induced by acidosis and recovery are shown. *Acidosis vs recovery was significant (P<0.01). (c) Distribution of AE1 expression in α-intercalated cells. Left panels illustrate how the AE1 distribution was measured using the line function of the ImageJ software (see Materials and Methods), and the middle panels show representative plots of densitometric values along lines bisecting the respective α-intercalated cells. The right panels show a plot of the percentage of the total number of AE1-expressing cells that fall into basolateral/apical ratio bins ranging from 2 (predominant basolateral AE1 expression). (d) Frequency of AE1 and aquaporin-2 (AQP2)-positive cells per 100 μm length of cortical collecting duct (CCD) or outer medullary collecting duct (OMCD). Length was measured with the ImageJ software using kidney sections from two (7-day acidosis) or three (all others) rabbits per condition, and the corresponding number of AE1- or AQP2-positive cells observed in 88–373 CCDs and 69–172 OMCDs per condition was recorded (see Table 2). Results are presented as the average±s.e. for AE1-positive cells per 100 μm. *Normal vs acidotic (3 day), and **acidotic (3 day) vs recovery were significant (P<0.02).View Large Image Figure ViewerDownload (PPT) Abbreviations: AE1, anion exchanger; CCD, cortical collecting duct; magn., magnification. Abbreviations: Acid., acidosis; AE1, anion exchanger; AQP2, aquaporin 2; CD, collecting duct. Amelioration of acidosis by alkali loading reversed adaptive changes in pendrin expression. Pendrin staining intensity returned to near-normal levels (1.9-fold increase over acidosis staining intensity) in ‘recovery’ animals, whereas the pendrin cap size showed an intermediate distribution between that found in normal and acidotic animals (Figure 2b and c). The mean pendrin cap areas in μm2 were normal=35±2.1, acidotic =20.7±5.2, and recovery=32±0.6, indicating a restoration of apical pendrin expression within 12–18 h after administration of sodium bicarbonate. The apparent number of pendrin-positive cells also returned to normal upon amelioration of acidosis. These results suggest that the subcellular distribution of pendrin, as well as pendrin expression levels, continuously (and relatively rapidly) vary with changes in acid/base status. Adaptive changes in α-intercalated cells caused by fluctuation in acid/base status was also examined by immunofluorescence staining of kidney sections for AE1 (Table 1). Acidosis induced a reversible redistribution of AE1 to the basolateral membrane of α-intercalated cells located in the outer cortex (Figure 3a). As shown in Figure 3b, acidosis nearly doubled (1.7±0.1 times) the intensity of basolateral AE1 staining in the CCD, and this increase was reversed by administration of alkali to acidotic rabbits. Redistribution of AE1 was also quantified by measuring the intensity of AE1 staining along the vertical axis of the cell with the ImageJ software (US National Institutes of Health, Bethesda, MD, USA), as illustrated in Figure 3c (left panel), and calculating the ratio of the basolateral/apical peak intensity values determined from a plot of intensity versus position (Figure 3c, middle panels), as described in the Materials and Methods. A histogram of the proportion of cells with basolateral/apical ratios ranging from 2 (Figure 3c; right panels) reveals that acidosis markedly increased the proportion of cells with predominantly basolateral distribution of AE1 (that is, ratio of >1.4) compared with normal animals in which most cells showed ratios of 1.4 was: normal=19.7±8.8, acidotic=56.9±4.9, and recovery=16.3±3.9. Recovery from acidosis (alkali loading) resulted in an AE1 distribution pattern similar to that observed in normal animals. The acidosis-induced basolateral AE1 redistribution most likely reflects exocytosis into the basolateral membrane of α-intercalated cells, and thus AE1 cellular trafficking is continuously regulated by fluctuations in acid/base status. Acidosis over a 3- to 7-day period also resulted in increase in the apparent number of AE1+ and aquaporin 2 (AQP2)-positive cells in the collecting duct. The apparent frequency of AE1+ and AQP2-positive cells in the CCD increased 1–2 and 2–4 cells per 100 μm, respectively, during acidosis (Figure 3d). Similar increases in the number of AE1+ and AQP2-positive cells with acidosis were observed in the outer medullary collecting duct (OMCD), indicating that the apparent gain in AE1-positive cells does not necessarily result from conversion of β- to α-intercalated cell phenotype. Furthermore, efforts to define a pendrin/AE1 double-positive cell population in the rabbit CCD during acidosis failed to yield definitive results (not shown). Recovery from acidosis returned the apparent number of AE1 and AQP2 cells to near-normal levels, showing the rapid reversibility of these phenomena. Changes in intercalated and principal cell numbers reported in Figures 2 and 3d most likely reflect changes in transporter expression over and under a threshold for immunofluorescence detection. To determine whether changes in AE1 and pendrin staining intensity reflect regulation of transporter gene expression by acid/base status, the steady-state levels of AE1 and pendrin mRNAs expressed in rabbit kidney cortex were determined by quantitative real-time PCR. As shown in Figure 4 (top panel), the steady-state level of pendrin mRNA in kidney cortex isolated from normal rabbits is 131-fold greater than the steady-state level of AE1 mRNA, due in part to the fact that β-intercalated cells outnumber α-intercalated cells by more than 3:1 in the normal rabbit CCD (compare Figures 2d and 3d).1.Schwartz G.J. Barasch J. Al-Awqati Q. Plasticity of functional epithelial polarity.Nature. 1985; 318: 368-371Crossref PubMed Scopus (243) Google Scholar, 19.Satlin L.M. Matsumoto T. Schwartz G.J. Postnatal maturation of rabbit renal collecting duct. III. Peanut lectin-binding intercalated cells.Am J Physiol. 1992; 262: F199-F208PubMed Google Scholar, 20.Matsumoto T. Fejes-Toth G. Schwartz G.J. Postnatal differentiation of rabbit collecting duct intercalated cells.Pediatr Res. 1996; 39: 1-12Crossref PubMed Scopus (18) Google Scholar, 21.Matsumoto T. Schwartz G.J. Novel method for performing carbonic anhydrase histochemistry and immunocytochemistry on cryosections.J Histochem Cytochem. 1992; 40: 1223-1227Crossref PubMed Scopus (12) Google Scholar Pendrin mRNA expression in kidney cortex from acidotic rabbits was reduced 3.0-fold compared with normal rabbits (P 0.5). After 3 days of acid loading, pendrin mRNA abundance was still 46-fold greater than AE1. Amelioration of acidosis by alkali loading reversed the effect of acidosis on pendrin mRNA expression; pendrin mRNA abundance returned to essentially normal levels in ‘recovery’ animals (normal vs recovery; P>0.5). In a previous study, steady-state levels of epithelial sodium channel (ENaC) β-subunit mRNA analyzed by Northern analysis did not seem to be altered by acidosis.22.Faroqui S. Sheriff S. Amlal H. Metabolic acidosis has dual effects on sodium handling by rat kidney.Am J Physiol Renal Physiol. 2006; 291: F322-F331Crossref PubMed Scopus (26) Google Scholar Therefore, we examined ENaC β-subunit copy number as a possible control for the relative amount of distal nephron segments contained in outer cortex samples. However, ENaC β copy number was reduced 1.5-fold (P 0.2) upon amelioration of acidosis. As B1-V-ATPase mRNA did not change during acidosis (Figure 4, upper panel), normalization to B1-V-ATPase copy number was used to control for the relative amount of distal nephron segments in RNA from kidney cortex. The B1-V-ATPase copy number from a normal rabbit kidney cortex sample was assigned the value of 1 (that is, division by itself), and then the pendrin and AE1 copy numbers were adjusted for the difference in the B1 mRNA abundance between the control and the respective sample. As shown in Figure 4 (lower panel), even with normalization to B1-V-ATPase, acidosis did not result in a significant change in AE1 mRNA abundance (P>0.15). The reduction in pendrin mRNA abundance by acidosis was 3.1-fold after normalization (P 0.5). These results suggest that adaptive changes in pendrin expression observed in Figure 2 are due, at least in part, to regulation of pendrin gene expression by acid/base status. To confirm regulation of pendrin expression by acid/base status at the protein level, we prepared membrane vesicles from rabbit kidney cortex and quantified levels of pendrin, AE1, and B1-V-ATPase protein by western blotting. As intercalated cells represent only a small fraction of the total epithelial cell population in the rabbit kidney cortex, we performed Percoll density gradient fractionation of membrane vesicle preparations to enrich for vesicle fractions containing acid/base transporters. This approach enhanced detection of all transporters, but was essential for detection of AE1 protein in the kidney cortex. As shown in Figure 5a, pendrin was detected as a major band between 110 and 140 kDa as well as a purported dimeric form of ∼220–250 kDa (not shown) that distributed roughly evenly across the density gradient in vesicle preparations from normal rabbits, whereas AE1 was detected in low fractions as a major band of ∼230 kDa (presumably a glycosylated dimeric form) as well as ∼100–110 kDa species (glycosylated monomeric form); deglycosylation reduced the size to ∼90 kDa (not shown). Consistent with the results for mRNA abundance, acidosis reduced the overall level of pendrin protein in gradient fractions (that is, sum of levels in individual fractions) by approximately threefold (normal vs acidosis: P<0.035; acidosis vs recovery: P 0.5 for AE1 and B1). Occasionally, as in the experiment shown in Figure 5a, there was an apparent shift in the pendrin distribution from low-density (fraction ≥8 in normals) to high-density fractions (F4 in acidosis) that may reflect changes in pendrin trafficking into distinct vesicular compartments during acidosis.17.Wagner C.A. Finberg K.E. Stehberger P.A. et al.Regulation of the expression of the Cl-/anion exchanger pendrin in mouse kidney by acid-base status.Kidney Int. 2002; 62: 2109-2117Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar,23.Kim Y.H. Kwon T.-H. Frische S. et al.Immunocytochemical localization of pendrin in intercalated cell subtypes in rat and mouse kidney.Am J Physiol Renal Physiol. 2002; 283: F744-F754Crossref PubMed Scopus (154) Google Scholar Furthermore, redistribution of pendrin to lower-density fractions upon recovery was consistently observed. Recovery from acidosis restored pendrin protein expression to essentially normal levels (normal vs recovery; P>0.5). Figure 5c illustrates the close association between urine pH and pendrin protein expression (r2=0.827), and provides additional evidence for rapid and reversible regulation of pendrin protein expression by acid/base perturbations. In a recent study, Van Huyen et al.24.Van Huyen J.P. Cheval L. Bloch-Faure M. et al.GDF15 triggers homeostatic proliferation of acid-secreting collecting duct cells.J Am Soc Nephrol. 2008; 19: 1965-1974Crossref PubMed Scopus (56) Google Scholar observed proliferation of α-intercalated cells, particularly in the OMCD that peake