Regulated expression of pendrin in rat kidney in response tochronic NH4Cl or Na - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:32 |只看该作者 |倒序浏览 |打印

作者：SebastianFrische, Tae-HwanKwon, JørgenFrøkiær, Kirsten M.Madsen,  SørenNielsen,作者单位：1 The Water and Salt Research Center, Institute ofAnatomy, and Institute of Experimental Clinical Research,University of Aarhus, DK-8000 Aarhus C, Denmark; Departmentof Physiology, School of Medicine, Dongguk University, 780-714 Kyungju, Korea; and Department of Medicine, University ofFlorida, Gaine
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      【摘要】! R2 ^' v5 _& y& O1 T9 e) u
   The anionexchanger pendrin is present in the apical plasma membrane of type Band non-A-non-B intercalated cells of the cortical collecting duct(CCD) and connecting tubule and is involved in HCO 3 − secretion. In this study, we investigated whether the abundance andsubcellular localization of pendrin are regulated in response toexperimental metabolic acidosis and alkalosis with maintained water andsodium intake. NH 4 Cl loading (0.033 mmolNH 4 Cl/g body wt for 7 days) dramatically reduced pendrin abundance to 22 ± 4% of control values ( n = 6, P labeling for pendrinshowed reduced intensity in NH 4 Cl-loaded animals comparedwith control animals. Moreover, double-label laser confocal microscopyrevealed a reduction in the fraction of cells in the CCD exhibitingpendrin labeling to 65% of the control value ( n = 6, P 3 loading(0.033 mmol NaHCO 3 /g body wt for 7 days) induced asignificant increase in pendrin expression to 153 ± 11% ofcontrol values ( n = 6, P Immunoelectronmicroscopy revealed no major changes in the subcellular distribution,with abundant labeling in both the apical plasma membrane and theintracellular vesicles in all conditions. These results indicate thatchanges in pendrin protein expression play a key role in thewell-established regulation of HCO 3 − secretion in theCCD in response to chronic changes in acid-base balance and suggestthat regulation of pendrin expression may be clinically important inthe correction of acid-base disturbances.
      【关键词】 collecting duct acidbase balance intercalated cells electronmicroscopy immunocytochemistry
   INTRODUCTION0 L# J' j& o8 I. f" v+ H
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THE INTERCALATED CELLS OF the collecting duct play an important role in acid-baseregulation in the mammalian kidney. On the basis of both themorphological characteristics ( 30 ) and the subcellularlocalization of acid-base transporters ( 2, 7, 14 ), twomain types of intercalated cells, type A and type B, can bedistinguished in the cortical collecting duct (CCD) and connectingtubule (CNT).
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Type A intercalated cells secrete protons into the urine and reabsorbHCO 3 − to the blood ( 23 ). They exhibit thevacuolar proton pump in the apical plasma membrane and apical vesiclesand the kidney splice variant of anion exchanger 1 (AE1) in thebasolateral plasma membrane ( 2 ).

Type B intercalated cells operate in the reverse mode. They secreteHCO 3 − in exchange for Cl across theapical membrane ( 17, 23 ), whereas protons are secreted tothe systemic circulation by the vacuolar type H -ATPaselocalized in the basolateral membrane. Recent studies have demonstratedthat the anion exchanger pendrin is present in the apical domain oftype B intercalated cells in the CCD of both rat and mouse kidney( 19 ). Moreover, a comparison between pendrin-deficient andwild-type mice revealed that pendrin is essential forHCO 3 − secretion in the CCD after NaHCO 3 loading ( 19 ). Pendrin has also been shown to be expressedin human kidney ( 15 ).7 u. I8 v7 @9 O! r1 F9 y2 N3 Z4 D

A third type of intercalated cell, non-A-non-B ( 12 ), ispresent in low numbers in the CCD and CNT of rats but is abundant inmouse CNT ( 10 ). The non-A-non-B type intercalated cellsexhibit vacuolar proton pumps as well as pendrin ( 10, 13 )in the apical plasma membrane and no basolateral AE1 ( 10 ).The function of non-A-non-B type intercalated cells has not beeninvestigated; accordingly, the role of pendrin in these cells remains unclear.

The rat CCD is capable of either net HCO 3 − secretionor net HCO 3 − absorption, depending on the systemicacid-base status ( 4 ). CCD segments from 24-hNH 4 Cl-loaded rats absorb HCO 3 −, and CCDfrom 24-h NaHCO 3 -loaded rats secrete HCO 3 − ( 4 ). In CCD from rats made acutely alkalotic bydeoxycorticosterone injections ( 16 ) or peritoneal dialysisagainst an NaHCO 3 solution ( 8 ),HCO 3 − secretion is increased and is dependent onluminal Cl. Furthermore, CCD from fasted rats absorbHCO 3 −, but this is reversed to secretion if the CCDare perfused with 100 mM Cl ( 16 ). ThusHCO 3 − secretion in the rat CCD is regulated inresponse to changes in systemic acid-base status. However, themolecular and cellular/subcellular events that are responsible for thefunctional regulation of HCO 3 − secretion remain to be established.

Previous studies have described the cellular response to acid-basedisturbances in the rat CCD. In type A intercalated cells, the apicalmembrane area is increased in response to respiratory acidosis( 30 ). This has been interpreted as the result of vesicle trafficking to the apical plasma membrane increasing the number ofactive proton pumps in the apical plasma membrane in response toacidosis ( 6 ). In contrast, acute metabolic alkalosisresults in a reduced apical membrane area of type A intercalated cells in rat CCD and reduced density of H -ATPase in the apicalplasma membrane ( 29 ). These and other observations( 6 ) suggest that acute changes in proton andHCO 3 − transport in type A intercalated cells in ratsare, at least in part, mediated by changes in trafficking ofH -ATPase between the apical plasma membrane andintracellular tubular vesicles and changes in AE1 synthesis ( 9, 20, 27 ).) ?1 V6 |/ {; b* o7 r
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Less is known about the response of type B intercalated cells in therat to acid-base disturbances. The morphology of type B intercalatedcells does not change during acute respiratory acidosis( 30 ), but in rats subjected to acute metabolic alkalosis the type B intercalated cells are larger and show an enlarged basolateral membrane area ( 29 ). However, there were nochanges in the subcellular localization of H -ATPase intype B intercalated cells during acute metabolic alkalosis ( 29 ).

The recent demonstration that pendrin is present in the apical plasmamembrane of type B intercalated cells and plays a role inHCO 3 − secretion in the CCD ( 19 ) raisesthe question of whether the functional regulation ofHCO 3 − secretion in response to acid-base disturbancesis mediated by changes in pendrin expression/activity. Therefore, thepurpose of the present study was to investigate whether the abundance and subcellular localization of pendrin are regulated or altered inresponse to metabolic alkalosis and acidosis induced by chronic NaHCO 3 or NH 4 Cl loading, respectively. This wasachieved by semiquantitative immunoblotting, immunohistochemistry,confocal laser microscopy, and immunoelectron microscopy.1 V. ], c4 X. G% G& e& I8 P
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MATERIALS AND METHODS
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Animals
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Male Munich-Wistar rats (250-300 g) from MøllegaardBreeding Centre were kept on a standard rodent diet (Altromin, Lage,Germany) until the experimental protocol was started. Rats wereassigned randomly to either the control or the treated groups. Beforetreatment, the rats were kept in metabolic cages for 3 days to recordbaseline values.

NH 4 Cl and NaHCO 3 Loading with FixedWater and Food Intake' A5 f" b0 E7 S1 K' X* M+ x4 p/ h
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Two experimental protocols were used as previously described( 18 ).
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Protocol 1: NH 4 Cl loading. Each morning, the rats were given a fixed amount of ground rat food(0.068 g/g body wt) mixed with water (0.168 g/g body wt). Theexperimental group ( n = 12) was given 0.033 mmolNH 4 Cl/g body wt in the food for 7 days, whereas the controlgroup ( n = 12) received the same diet but withoutNH 4 Cl.
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Protocol 2: NaHCO 3 loading. Each morning, the rats were given a fixed amount of ground rat food(0.068 g/g body wt) mixed with water (0.168 g/g body wt). Theexperimental group ( n = 12) was given 0.033 mmolNaHCO 3 /g body wt in the food for 7 days, whereas thecontrol group ( n = 12) was given 0.033 mmol NaCl/g bodywt to balance sodium intake.
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Urine and Blood Sampling and Analysis) g. @6 F0 ?5 v) k6 o; u

Urine was collected every morning while the rats were inmetabolic cages. The pH of urine from the last 24 h beforetermination of the experiment was measured with a PHM83 pH meter(Radiometer, Copenhagen, Denmark), and osmolality was measured with anautomatic cryoscopic osmometer (Omomat 030, Gonotech, Berlin, Germany). Venous blood was drawn in gas-tight syringes from the vena cava beforeremoval of the kidneys used for immunoblotting. One aliquot of theblood sample was used immediately for blood-gas analysis with an ABLsystem 615 (Radiometer). The remaining blood was centrifuged for 15 minat 4,000 g to remove the blood cells, and subsequently theplasma was analyzed for sodium, potassium, and creatinine with a Vitros950 (Johnson & Johnson) and osmolality was measured with an automaticcryoscopic osmometer (Omomat 030, Gonotech).

Membrane Fractionation and Immunoblotting8 Z# I' H. {) W9 g+ W1 r
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Kidneys from 6 rats from each experimental group were used.Tissue from the cortex/outer stripe of the outer medulla washomogenized in 0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin and 1 mM phenylmethyl sulfonylfluoride,by using an ultraturrax T8 homogenizer (IKA Labortechnik) at maximumspeed for 30 s, and the homogenates were centrifuged in anEppendorf centrifuge at 4,000 g for 15 min at 4°C toremove whole cells, nuclei, and mitochondria. The supernatantwas then centrifuged at 200,000 g for 1 h to produce apellet containing membrane fractions enriched for both plasma membranesand intracellular vesicles. The samples were prepared for gelelectrophoresis by adding Laemmli sample buffer containing 2% SDS(final concentration) to the resuspended pellets.

Antibodies
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Polyclonal antibodies raised against a synthetic peptidecorresponding to 22 amino acids, MEAEMNAEELDVQDEAMRRLAS, of the COOH terminal of mouse pendrin were used to identify pendrin as previously described ( 13 ). A monoclonal antibody against ratCalbindin (RDI-CALBINDabm, Research Diagnostics) was used todistinguish CNTs from collecting ducts in sections of paraffin-embeddedrat kidney.

Electrophoresis and Immunoblotting  _; A5 }- U4 K0 u

Samples of rat kidney membranes (see MembraneFractionation and Immunoblotting for details) were loaded on 9%polyacrylamide minigels (Mini Protean II, Bio-Rad) and run for 1.5 h at 130 V. After transfer by electroelution (100 V, 1 h) tonitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na 2 HPO 4, 20 mMNaH 2 PO 4, 100 mM NaCl, and 0.1% Tween 20, pH7.5) for 1 h, and incubated overnight at 4°C with anti-pendrinantibodies. The labeling was visualized with horseradishperoxidase-conjugated secondary antibodies (diluted 1:3,000; P448,DAKO, Glostrup, Denmark) by using an enhanced chemiluminescence system(Amersham International). The chemiluminescence was recorded on film,which was subsequently scanned with a flatbed scanner. Densitometry wasperformed by using a custom-made computer program, Easy-Gel (DavidMarples, University of Leeds, Leeds, UK, unpublished)., A/ j, g( T8 h7 w
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Immunohistochemistry
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Kidneys from 6 rats from each experimental group were fixed byretrograde perfusion via the aorta with 4% paraformaldehyde in 0.1 Mcacodylate buffer, pH 7.4, and postfixed for 2 h in the samefixative. Kidney slices containing all kidney zones were dehydrated andembedded in paraffin. The paraffin-embedded tissues were cut at 2 µmon a rotary microtome (Leica, Heidelberg, Germany). The sections weredewaxed and rehydrated. To reveal antigens, sections were placed in 1 mM Tris buffer (pH 9.0) supplemented with 0.5 mM EGTA and heated in amicrowave oven for 10 min. Nonspecific binding of Ig was prevented byincubating the sections in 50 mM NH 4 Cl for 30 min followedby blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2%gelatin. Sections were incubated overnight at 4°C with pendrinantibodies diluted in 10 mM PBS, pH 7.4, containing 0.1% Triton X-100and 0.1% BSA. For light microscopy, sections were incubated withhorseradish peroxidase-linked goat anti-rabbit secondary antibodies(P448, DAKO, Glostrup, Denmark), labeling was visualized bydiaminobenzidine technique, and the sections were counterstained withMayers hematoxylin. For laser confocal microscopy,calbindin was localized with mouse monoclonal antibodies that weremixed with the antibody against pendrin. The labeling was visualizedwith an Alexa 546-conjugated goat anti-mouse antibody (diluted 1:200;Molecular Probes) mixed with an Alexa 488-conjugated goat anti-rabbitantibody (diluted 1:200; Molecular Probes). Confocal laser microscopywas carried out with a Leica SP2 laser confocal microscope.

Cell Counting

To evaluate whether the fractions of cells in CNT and CCDshowing immunoreactivity for pendrin were changed in acidosis and alkalosis, sections labeled for calbindin and pendrin were analyzed asfollows. First, cross sections of CNT were identified as tubules withlabeling for both calbindin and pendrin, and cross sections of CCD wereidentified as tubules with labeling for pendrin only. Second, theclearly defined nuclei in the identified tubules were counted by usinga differential interference contract (DIC) image obtained concomitantlywith the fluorescence images. Third, the nuclei pertaining to cellsthat also labeled for pendrin were counted. One kidney section fromeach of five NH 4 Cl-loaded and five control rats and fromeach of four NaHCO 3 -loaded and three control rats wasinspected. In each section, at least five cross sections of CNT andfive cross sections of CCD were identified, and at least 62 cells werecounted from each tubule segment in each animal. In total 1,943 cellswere counted. The fraction of pendrin-labeled cells was calculated asthe number of nuclei in pendrin-positive cells found in one animaldivided by the total number of nuclei counted in this animal. Thisprocedure underestimates the total number of cells in CNT and CCD,because tubular cross sections devoid of pendrin labeling were notcounted. Therefore, the absolute fraction of pendrin-positive cells isoverestimated. However, the measurements are only intended forcomparison within this study, enabling a semiquantitativeinterpretation of the labeling patterns between treated and control rats.$ T1 I# L. R- P" V) u9 Z

Before averaging, normalization with respect to control values andfurther statistical analysis, the fraction scale data were arc-sintransformed to obtain normality ( 34 ).8 u1 A% h% |- Z
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Immunoelectron Microscopy
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For immunoelectron microscopy, small pieces of kidney cortexwere cut from slices of fixed kidney (see Immunohistochemistry ), cryoprotected in 2.3 M sucrose, andfrozen in liquid nitrogen. The frozen samples werefreeze-substituted in a Reichert AFS freeze substitution unit. Inbrief, the samples were sequentially equilibrated over 3 days inmethanol containing 0.5% uranyl acetate at temperatures graduallyraised from 80 to 70°C, rinsed in pure methanol for 24 hwhile increasing the temperature from 70 to 45°C, and infiltrated with graded Lowicryl HM20 and methanol solutions (1:1, 2:1) and pureLowicryl HM20 before UV polymerization for 2 days at 45°C and 2 days at 0°C. Immunolabeling was performed on ultrathin Lowicryl HM20sections. Sections were pretreated with the saturated solution of NaOHin absolute ethanol (2-3 s), rinsed, and preincubated for 10 minwith 0.1% sodium borohydride and 50 mM glycine in 0.05 M Tris, pH 7.4, containing 0.1% Triton-X 100. Sections were rinsed and incubatedovernight at 4°C with primary antibodies diluted in 0.05 M Tris, pH7.4, containing 0.1% Triton-X 100 with 0.2% milk (diluted 1:200).After being rinsed, sections were incubated for 1 h at roomtemperature with goat anti-rabbit IgG conjugated to 10-nm colloidalgold particles (1:50; GAR.EM10, BioCell Research Laboratories, Cardiff,UK). The sections were stained with uranyl acetate and lead citratebefore examination in a Philips Morgagni electron microscope operatingat 70 kV.5 w( K4 l& p9 w- L

RESULTS

NH 4 Cl-Loaded Rats Showed Reduced Urine pH, DecreasedPlasma Sodium, Increased Plasma Potassium, and Increased UrineOsmolality

The urine and blood acid-base parameters of theNH 4 Cl-loaded and control groups are shown in Table 1. Urine proton concentration differedsignificantly between the experimental group and the control group;accordingly, marked differences in urine pH were seen (5.76 vs. 7.94).Blood acid-base parameters (plasma [H ] and plasma[HCO 3 − ], where brackets indicate concentration,plasma total CO 2, and plasma PCO 2 ) were unchanged between experimental and control groups." L0 e' G: p% x+ J- W
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Table 1. Blood and urine data

Pendrin Abundance in NH 4 Cl-Loaded Rats Was MarkedlyReduced

Semiquantitative immunoblotting of 4,000- g supernatantsof homogenized rat kidney cortex and outer stripe of the outer medulla from NH 4 Cl-loaded rats showed a marked reduction in theamount of detectable pendrin: 22 ± 4 vs. 100 ± 11%, P 1, A and B ). Similarly, immunoperoxidaselabeling for pendrin in sections of paraffin-embedded kidneys fromNH 4 Cl-loaded rats showed less intense staining in the outercortex (CNT and CCD segments) than sections from control rats whenanalyzed at low magnification (Fig. 2, A and B ). Kidney sections from 6 NH 4 Cl-loaded rats and 5 control rats were inspected andshowed patterns consistent with the examples shown." r$ T; J) h: `8 A. M' I

Fig. 1. Semiquantitative immunoblottting of 4,000- g supernatant of homogenized rat kidney tissue from the cortex and outerstripe of the outer medulla obtained from NH 4 Cl-loaded andcontrol rats. A : immunoblot of samples from 6 NH 4 Cl-loaded and 6 control rats incubated with anti-pendrinantibody showing a distinct band at ~127 kD. B :densitometric analysis showed that the abundance of pendrin inNH 4 Cl-loaded rats was reduced to 22 ± 4% of controlvalues (100 ± 11%). * P! Y! ~, C5 i% b$ C- {- x

Fig. 2. Micrographs showing immunocytochemical localization of pendrin insections of paraffin-embedded kidneys from NH 4 -loaded rats( A and C ) and control rats ( B and D ). At low magnification, the labeling inNH 4 -loaded rats ( A ) appeared less abundant thanin control rats ( B ). At high magnification, pendrin labelingwas seen in the apical part of intercalated cells in connecting tubules(CNT) and cortical collecting ducts (CCD) in bothNH 4 -loaded rats ( C ) and control rats( D ). P, proximal tubule; G, glomerulus; arrows, cellsshowing immunoreactivity for pendrin./ X- U. T4 z$ V0 I& K

Fraction of CDD and CNT Cells with Pendrin Immunoreactivity WasReduced in NH 4 Cl-Loaded Rats5 m( y4 ]3 _) c7 @* t
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At higher magnification, the reduced labeling of pendrin was alsoapparent, although the difference in the intensity of labeling at thelevel of individual CCD and CNT profiles and intercalated cells wasless pronounced (Fig. 2, C and D ). Both CNT andCCD segments showed reduced labeling. Furthermore, it appeared that thenumber of cells exhibiting pendrin labeling was markedly reduced inresponse to NH 4 Cl loading (Fig. 2, C and D ). To examine this further, laser confocal and DICmicroscopy were performed by using double-immunolabeled (for pendrinand calbindin) sections of paraffin-embedded kidneys. To evaluatewhether the fraction of cells with detectable pendrin immunoreactivitywas changed, pendrin-labeled cells and the total number of cells incross-sectioned tubules with pendrin-labeled cells in CNT and CCD werecounted. To illustrate the counting procedure, an example of the imagesused for cell counting is shown in Fig. 3, A-D. The resultsrevealed that the fraction of cells exhibiting pendrin immunoreactivityin CCD was significantly reduced in NH 4 Cl-loaded ratscompared with control values (65 ± 4% of the control value, P 2; Fig. 3 E ). In CNT, the fraction of cells exhibiting pendrinimmunoreactivity was not significantly different from the control value(87 ± 5% of control value, P = 0.26; Table 2;Fig. 3 ).# o2 d$ [5 X- Y; N) I

Fig. 3. The fraction of epithelial cells in rat CCD thatexhibited immunocytochemical labeling for pendrin was markedly reducedin NH 4 -loaded rats. A-D : laser confocalmicroscopy of a section of paraffin-embedded rat kidney. These andsimilar sets of images were used for counting cells and calculating thefraction of pendrin-labeled epithelial cells in CCD and CNT asdescribed in the text. A : red labeling of calbindin. B : green labeling of pendrin. C : differentialinterference contrast image generated from the transmitted laser lightat 488 nm. D : overlay of A - C.Note the disjunctive distribution of calbindin associated with CNTcells and pendrin associated with type B intercalated cells. Whitedots, clearly defined nuclei; white dots encircled with black ring,nuclei in cells, which label for pendrin. E : fraction ofepithelial cells in rat CCD that exhibited immunocytochemical labelingfor pendrin was reduced to 65% of the control value inNH 4 Cl-loaded rats, whereas the reduction to 87% of thecontrol value in CNT was statistically insignificant.* P

Table 2. Cells counted in CNT and CCD

Subcellular Localization of Pendrin Was Not Changed in Response toChronic NH 4 Cl Loading

Immunoelectron microscopical analysis of pendrinlocalization confirmed reduced immunogold labeling inNH 4 Cl-loaded rats compared with control animals. However,there were no apparent differences in the subcellular localization ofpendrin between NH 4 Cl-loaded and control rats (Fig. 4, A and B ). Inboth groups, pendrin was localized at the apical plasma membrane and inintracellular vesicular structures in the apical part of type Bintercalated cells.6 X) ]1 r& m; o& s* Q
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Fig. 4. Immunoelectron microscopic localization of pendrin in CCD ofNH 4 Cl-loaded ( A ) and control rats( B ). Pendrin labeling was observed in the apical plasmamembrane and in intracellular vesicles of intercalated cells in bothNH 4 Cl-loaded rats ( A ) and control rats( B ). Arrows, immunogold-labeled pendrin.

NaHCO 3 -Loaded Rats Showed Increased Urine pH" s* Z3 C: B6 I! w# T6 `0 t

The urine and blood acid-base parameters of theNaHCO 3 -loaded and control groups are shown in Table 1.Urine proton concentration differed significantly between theexperimental group and the control group; accordingly, markeddifferences in urine pH were seen (8.77 vs. 7.40). Blood acid-baseparameters (plasma [H ], plasma[HCO 3 − ], plasma total CO 2, and plasmaPCO 2 ) were unchanged between the NaHCO 3 -loadedand control group.
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Pendrin Abundance in NaHCO 3 -Loaded Rats Was MarkedlyIncreased+ J+ W/ o" [& _7 z& }& O# X
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Semiquantitative immunoblotting of 4,000- g supernatants of homogenized rat kidney cortex and outer stripe of theouter medulla from NaHCO 3 -loaded rats showed a significantincrease in the amount of detectable pendrin: 153 ± 11 vs.100 ± 12%, P 5, A and B ).Consistent with this, immunoperoxidase-labeled sections ofparaffin-embedded kidneys from NaHCO 3 -loaded rats (Fig. 6, A and C )exhibited an increase in the intensity of pendrin immunostaining compared with sections from control rats (Fig. 6, B and D ). The increase in labeling intensity was equallydistributed over the labeled cells, i.e., the change was not onlyobserved in a subset of cells. Kidney sections from fourNaHCO 3 -loaded rats and three NaHCO 3 -controlrats were inspected.

Fig. 5. Semiquantitative immunoblottting of 4,000- g supernatant of homogenized kidney tissue from the cortex and outerstripe of the outer medulla obtained from NaHCO 3 -loaded andcontrol rats. A : immunoblot of samples from 6 NaHCO 3 -loaded and 6 control rats incubated withanti-pendrin antibody showing a distinct band at ~127 kD. B : densitometric analysis showed that the abundance ofpendrin in NaHCO 3 -loaded rats increased to 153 ± 11%of control values (100 ± 12%). * P5 B" i5 `+ n& W6 U* g) l4 p7 l  K8 e7 n
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Fig. 6. Micrographs showing immunocytochemical localization of pendrin insections of paraffin-embedded kidneys from NaHCO 3 -loadedrats ( A and C ) and control rats ( B and D ). A and B : at low magnification,pendrin labeling was observed in CCD and CNT. Limited increasedlabeling was observed in response to NaHCO 3 loading,but no apparent difference was seen in the overall labelingpattern between NaHCO 3 -loaded rats ( A ) andcontrol rats ( B ). At high magnification, pendrin labelingwas seen in the apical part of intercalated cells in CNT and CCD inboth NaHCO 3 -loaded rats ( C ) and control rats( D ). Arrows, immunoreactivity for pendrin.5 y: q" b# G# q! o$ T5 {4 O
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Fraction of CCD and CNT Cells with Pendrin Immunoreactivity WasUnchanged in NaHCO 3 -Loaded Rats

Laser confocal microscopy and DIC microscopy ofdouble-immunolabeled (for pendrin and calbindin) paraffin sectionsrevealed that the fraction of pendrin-labeled epithelial cells in CNTand CCD was unchanged in NaHCO 3 -loaded rats (CNT: 102 ± 4% of control values, P = 0.80; CCD: 112 ± 9% of control values, P = 0.41; Table 2; Fig. 7 ).0 K, O# @, p  Y/ {& K4 }7 X

Fig. 7. The fraction of epithelial cells in rat CNT and CCD thatexhibit immunocytochemical labeling for pendrin (determined bydouble-label laser scanning confocal microscopy) was unchanged betweencontrol rats and NaHCO 3 -loaded rats. Cells were counted asexplained in the text and illustrated in Fig. 3.
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Subcellular Localization of Pendrin Was Not Changed in Response toNaHCO 3 Loading

Electron microscopical investigation of immunolabeled kidneysections from NaHCO 3 -loaded rats (Fig. 8 A ) did not show consistent differences compared with control animals (Fig. 8 B )regarding the subcellular localization of pendrin. In both groups,pendrin was localized at the apical plasma membrane and inintracellular vesicular structures in the apical part of type Bintercalated cells. The relative distribution of labeling between theplasma membrane and the intracellular compartment varied from cell to cell in both groups, but no quantitative measures of this variation were obtained.$ |8 `: \& x# D" j+ z, T$ _
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Fig. 8. Immunoelectron microscopic localization of pendrin in CCD ofNaHCO 3 -loaded ( A ) and control rats( B ). Pendrin immunogold labeling was observed in the apicalplasma membrane and in intracellular vesicles of intercalated cells inboth the NaHCO 3 -loaded rat ( A ) and control rats( B ). Arrows, immunogold-labeled pendrin./ V7 ?/ K/ _; E+ M8 Q3 I0 v7 z1 p
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DISCUSSION

This study documents a marked reduction in pendrin abundancein the CCD and CNT of rats in response to chronic NH 4 Clloading. In contrast, chronic NaHCO 3 loading resulted in asignificant increase in pendrin abundance. These findings are inaccordance with results of previous studies ( 4, 8, 16, 33 )of HCO 3 − transport in the CCD and strongly indicatethat the regulation of HCO 3 − secretion in the rat CCDin response to acid-base disturbances involves changes in pendrin abundance in the type B intercalated cells. The results alsoprovide additional support for the importance of intercalated cells in the CCD and CNT in the regulation of systemic acid-base balance. On thebasis of these observations, we conclude that HCO 3 − secretion can be stimulated or inhibited through changes in pendrin abundance in intercalated cells in the CCD and CNT. Thus the regulation of pendrin expression in the collecting duct represents a molecular mechanism to control HCO 3 − secretion and correctacid-base disturbances.& ^4 e2 U6 O) v  y
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Pendrin Abundance Is Increased in NaHCO 3 -Loaded Ratsand Decreased in NH 4 Cl-Loaded Rats) e4 \2 i3 b0 w
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Because the increase in pendrin after NaHCO 3 loadingis not restricted to a subset of cells, it is most likely due to anincreased amount of pendrin in both intercalated type B cells andintercalated cells of the non-A-non-B type. Similarly, the reduction inpendrin seen by immunoblotting after NH 4 Cl loading cannotsolely be due to changes in non-A-non-B cells because of the lowfraction of these cells in rat CNT and CCD (5.9 and 2.1%,respectively) ( 10 ).

The changes in pendrin abundance demonstrated in this study are inagreement with the results of physiological studies indicating anincreased capacity for HCO 3 − secretion in CCD fromNaHCO 3 -loaded animals and a reduced capacity forHCO 3 − secretion in NH 4 Cl-loaded animals( 4, 16 ).

The mechanism of HCO 3 − secretion in CCD has beenextensively studied. Star et al. ( 28 ) found that replacement of luminal and peritubular Cl with gluconatedecreases HCO 3 − secretion significantly, indicatingthat HCO 3 − secretion in CCD is dependent onCl. Subsequent studies by several laboratories haveconfirmed that HCO 3 − secretion in the CCD of both rats and rabbits is mediated by apicalCl /HCO 3 − exchange ( 23 ).Recent studies have demonstrated that the anion exchanger pendrin islocalized at the apical domains of HCO 3 − -secreting type B intercalated cells, and elegant studies using transgenic micelacking pendrin showed that pendrin is involved inHCO 3 − secretion ( 19 ). Subsequently,pendrin was shown also to be present in type non-A-non-B intercalatedcells ( 13 ). The present study indicates that regulation ofHCO 3 − secretion in CCD is, at least in part, dependenton changes in pendrin abundance in intercalated cells.
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The majority of filtered HCO 3 − is reabsorbed by theproximal tubule. Consistent with this, high rates of urinaryHCO 3 − excretion during alkali loading or in responseto metabolic alkalosis have largely been attributed to inhibition ofHCO 3 − reabsorption in the proximal tubule( 3 ). However, the final regulation of urine acidificationtakes place in the collecting duct, which is a site of bothreabsorption and secretion of HCO 3 −. On the basis ofthe results of this study, we propose that the fine control of urinaryHCO 3 − excretion is achieved by regulation ofHCO 3 − secretion through changes in pendrin abundancein the CNT and CCD. Thus when changes in HCO 3 − transport are required to achieve acid-base homeostasis, for example,in subchronic or chronic metabolic acidosis or alkalosis,HCO 3 − secretion can be inhibited or stimulated throughthe regulation of pendrin expression in the intercalated cells.

This mechanism may explain the results of a study of the effectof NaCl infusion on the correction of alkalosis induced by HCO 3 − loading. Here, it was found that the increase inurinary HCO 3 − secretion after NaCl infusion was muchhigher in alkalotic rats than in control rats ( 33 ). Wepropose that this may be related to an increased abundance of pendrinin HCO 3 − -loaded rats, as documented in the present study.# Y# ^& V: [9 h
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The regulatory mechanisms responsible for the changes in pendrinabundance are as yet unclear; however, a cAMP-dependent intracellular pathway is possibly involved, as indicated by the finding that cAMPincreases rabbit CCD HCO 3 − secretion ( 21 ). It has been proposed that the prostacyclin-inducedincrease in distal tubule HCO 3 − secretion acts throughthis pathway ( 31 ). Furthermore, a recent study hasindicated a possible role of endothelin-1 in regulating increaseddistal tubular acidification in response to acid ingestion( 32 ). Hypothetically, endothelin-1 could increase tubularacidification by reducing pendrin abundance in CCD type B intercalatedcells as found in NH 4 Cl-loaded animals in the present study.
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Fraction of CCD and CNT Cells Showing Pendrin Immunoreactivity WasReduced in NH 4 Cl-Loaded Rats, Whereas It Was Unchangedin NaHCO 3 -Loaded Rats- E% m, Y( a2 ^- m
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Controversy exists about whether the relative numbers of type Aand type B cells change during systemic acidosis ( 23 ). On the basis of in vitro studies in the CCD and in cultured collecting duct cells, it has been proposed that a reversal of polarity of type Bintercalated cells might account for the changes inH - ATPase labeling patterns observed in variousacid-base disturbances ( 1, 25, 26 ). However, the presenceof distinct anion exchangers in type A and type B intercalated cellsexcludes a simple relocation of the intercalated cell proteins as themechanism behind the observed changes. On the other hand, it ispossible that a more extensive epithelial remodeling might lead tochanges in the number of cells expressing pendrin and thus explain thechanges in the abundance of pendrin. To determine whether changes inacid-base status affect the fraction of pendrin-positive cells, wecompared estimates of the fractions of pendrin-labeled cells in CNT and CCD of NH 4 Cl-loaded, NaHCO 3 -loaded, and control animals.6 [% W. J* b2 U" o( D( g2 }
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As shown in Fig. 3 E, the reduced amount of pendrin inthe kidney cortex of NH 4 Cl-loaded rats was accompanied by asignificant reduction in the fraction of cells in the CCD that labelfor pendrin to 65% of control values. The reduction to 87% of controlvalues seen in CNT was not statistically significant but indicates that intercalated cells in the CNT may also loose pendrin immunoreactivity in response to NH 4 Cl loading, although to a lesser extentthan that in the CCD.

Pendrin immunoreactivity was never observed in the basolateral domain,so these results do not provide any evidence for a polarity change,i.e., relocation of the intercalated cell proteins. There are severalpossible explanations for the observed decrease in the fraction ofpendrin-positive cells in the CCD and CNT: 1 ) a decrease inpendrin abundance in individual cells leading to levels of expressionundetectable by immunohistochemistry; 2 ) a disappearance ofpendrin-expressing cells, e.g., by apoptosis or other forms ofcell deletion; and 3 ) a transformation of pendrin-expressing cells into other cell types, a scenario that would provide support forthe remodeling hypothesis previously proposed for type B intercalated cells. Distinguishing these three possible explanations for the reducedfraction of CCD and CNT cells exhibiting pendrin immunoreactivity isoutside the scope of the present study and awaits future investigations.7 f4 i" j& y2 D$ @$ [4 c% s. a
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It should be noted that support for the possibility of epithelialremodeling was provided in a recent study reporting a decrease in thepercentage of type B intercalated cells in the CCD of rats afterchronic treatment with acetazolamide, a carbonic anhydrase inhibitor,for 14 days ( 5 ). This decrease was associated with anincrease in the percentage of type A intercalated cells not only in theCCD but also in the collecting duct in the inner stripe of the outer medulla.
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Recent evidence from isolated perfused rabbit CCD indicates thatindividual type B intercalated cells lose apicalCl /HCO 3 − exchange activity after 3 h incubation at pH 6.8 ( 24 ). This suggests the presence ofa rapid mechanism for regulation of HCO 3 − secretion in type B intercalated cells consistent with previous experiments inisolated perfused CCD from rats ( 8, 16 ) and rabbits( 21 ). The disappearance of apicalCl /HCO 3 − exchange activity wasaccompanied by a reduction in apical plasma membrane area, which isindicative of endocytotic internalization ofCl /HCO 3 − exchangers ( 26 ),as previously proposed ( 8 ). We believe that irrespectiveof the cellular mechanisms, the previously observed reduction in thecapacity for HCO 3 − secretion by rat CCD in response tochronic NH 4 Cl loading is most likely due to a reduction in the abundance of active pendrin anion exchangers.

Interestingly, there were no significant changes in the fractions ofCCD and CNT cells showing pendrin immunoreactivity in response toNaHCO 3 loading. These observations suggest that the previously demonstrated increase in HCO 3 − secretionduring various alkalotic conditions is likely to be mediated by anincrease in pendrin expression/activity in already existing type Bintercalated cells rather than being associated with changes in thenumber of HCO 3 − -secreting cells.
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Subcellular Localization of Pendrin Is Unchanged inNH 4 Cl-Loaded and NaHCO 3 -Loaded Rats Comparedwith Control Rats
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The observed changes in the levels of expression of pendrin werenot accompanied by qualitative changes in the subcellular localizationof pendrin within the B-cells and non-A-non-B cells, as determined byimmunoelectron microscopy. Pendrin was found in the apical plasmamembrane and in intracellular vesicular structures in the apical partof intercalated type B and non-A-non-B cells inNH 4 Cl-loaded, NaHCO 3 -loaded, and two controlgroups. Thus there was no evidence that dramatic changes in traffickingor apical sorting of pendrin might be responsible for the regulation of CCD HCO 3 − secretion in response to chronic acid-basedisturbances. Because the animals in this study were subject to chronicacid and base loading, the subcellular localization of pendrin mostlikely represents steady-state situations in animals, which haveadapted to the constant load. This is supported by the observation thatplasma pH and total CO 2 levels were similar in all of theexperimental groups. Thus the present results do not preclude theoccurrence of transient short-term regulation involving trafficking ofpendrin molecules among cellular compartments in response to the acutedisturbances. Rather, it is quite possible that short-term acid-basedisturbances may induce rapid changes in the subcellular localizationof pendrin, which might explain the documented reduction incAMP-induced HCO 3 − secretion by rabbit CCD duringNH 4 Cl loading ( 21 ). The purpose of this studywas to evaluate the effect of chronic acid-base disturbances on pendrinexpression and localization; thus features pertaining to short-termregulation may have passed unnoticed. Whether insertion and removal ofpendrin molecules in the apical plasma membrane are responsible forrapid adjustments of HCO 3 − secretion in CCD should bepursued in future studies.

In conclusion, the present study demonstrates that pendrin proteinexpression in the kidney is tightly regulated in response to acid-basedisturbances and suggests that HCO 3 − secretion in theCCD and CNT is regulated through changes in pendrin abundance. Thuswhen changes in collecting duct HCO 3 − transport arerequired to achieve acid-base homeostasis, for example, in metabolicacidosis or alkalosis, HCO 3 − secretion can beinhibited or stimulated through the regulation of pendrin abundance inthe kidney.

ACKNOWLEDGEMENTS" m) g2 [9 V8 }) j# G+ j) n  S* n
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The authors thank Mette F. Vistisen, Lotte Vallentin Holbech, HelleHøyer, Zhila Nikrozi, Gitte Christensen, Merete Pedersen, and IngerMerete Paulsen for technical assistance. We thank the reviewers fortheir comments.# K, L7 A5 c4 A0 `' p! A6 d
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