Regulation of the apical Cl/HCO - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 22 下一页

返回列表

查看: 507498\|回复: 216	go Regulation of the apical Cl/HCO [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:25 |只看该作者 |倒序浏览 |打印

作者：SnezanaPetrovic, ZhaohuiWang, LiyunMa,  ManoocherSoleimani,作者单位：1 Department of Medicine, University of Cincinnati,and Veterans Affairs Medical Center at Cincinnati,Cincinnati, Ohio 45267-0485 # D6 H4 [2 A4 B! {* }
   ; v  h8 h; ^6 _

      % x- _( Y( k7 S, A
                     5 W- @' U: z" O) K
         0 D. o  t$ ?! o. T$ ~% R


         $ ^, w# g/ c1 n( [7 x




      【摘要】
   Pendrin is an apicalCl /OH /HCO 3 − exchanger in -intercalated cells ( -ICs) of rat and mouse cortical collectingduct (CCD). However, little is known about its regulation in acid-basedisorders. Here, we examined the regulation of pendrin in metabolicacidosis, a condition known to decrease HCO 3 − secretion in CCD. Rats were subjected to NH 4 Cl loading for4 days, which resulted in metabolic acidosis. ApicalCl /HCO 3 − exchanger activity in -ICswas determined as amplitude and rate of intracellular pH change when Clwas removed in isolated, microperfused CCDs. Intracellular pH wasmeasured by single-cell digital ratiometric imaging using fluorescentpH-sensitive dye2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein-AM. Pendrin mRNA expression in kidney cortex was examined by Northern blothybridizations. Expression of pendrin protein was assessed by indirectimmunofluorescence. Microperfused CCDs isolated from acidotic ratsdemonstrated ~60% reduction in apicalCl /HCO 3 − exchanger activity in -ICs( P the mRNA expression of pendrin in kidney cortexdecreased by 68% in acidotic animals ( P labeling demonstrated significantreduction in pendrin expression in CCDs of acidotic rats. We concludethat metabolic acidosis decreases the activity of the apicalCl /HCO 3 − exchanger in -ICs of the rat CCD by reducing the expression of pendrin. Adaptive downregulation ofpendrin in metabolic acidosis indicates the important role of thisexchanger in acid-base regulation in the CCD. 7 M! u* G+ D! x4 P* S$ X! |
      【关键词】 kidney intercalated cells
   INTRODUCTION
, Z# L) q2 [! _" r
ONE-THIRD OF THE KIDNEY CORTICAL collecting duct (CCD) cell population comprisesintercalated cells, which are responsible for the final adjustments ofacid-base balance ( 31 ). In response to acid or alkaliloading, CCD reabsorbs or secretes more HCO 3 −, respectively. This is accomplished through the coordinated activity oftwo types of intercalated cells: -intercalated cells ( -ICs), which are modeled to secrete acid through an apicalH -ATPase and basolateralCl /HCO 3 − exchanger, and -intercalated cells ( -ICs), which are modeled to secrete HCO 3 − through an apical Cl /HCO 3 − exchanger anda basolateral H -ATPase. Immunocytochemical experimentsidentified H -ATPase in both cell types as vacuolar-typeATPase and the basolateral Cl /HCO 3 − exchanger of -ICs as anion exchanger 1 (AE1) (reviewed in Ref. 31 ). However, the molecular identity of the apicalCl /HCO 3 − exchanger of -ICs has longremained unknown.9 E3 r/ N) a$ z" e6 h( m& @. d
% Q8 P( G) `" E% h* y
Recent molecular studies have identified a large, highly conservedfamily of membrane proteins (designated as SLC26A), many of which havebeen shown to transport anions. Three closely related members of thisfamily are downregulated in adenoma (DRA or SLC26A3), pendrin (PDS orSLC26A4), and PAT1 (CFEX or SLC26A6) ( 14, 17, 19, 20, 44 ).All three transporters mediate Cl /HCO 3 − exchange ( 22, 36, 44 ). DRA is expressed on the apicalmembranes of colonocytes, whereas PAT1 or CFEX is expressed on theapical membranes of kidney proximal tubule and duodenum ( 19, 22, 27, 44 ). Pendrin mRNA expression is detected in proximal tubuleand CCD ( 36 ). However, immunocytochemical studies localizependrin only to the apical membranes of a subpopulation of CCD cells,which also express H -ATPase on their basolateral membranes( 28, 35, 36 ). In addition, CCDs of pendrin-deficient micefailed to secret HCO 3 − in response toHCO 3 − loading ( 28 ). Taken together, thesestudies are consistent with pendrin functioning as an apicalCl /HCO 3 − exchanger in -ICs of CCD( 28, 35, 36 ).( j0 p2 n8 e: l/ y8 U4 R
" l# Y! X3 e3 {: g; S, M6 P2 ~# D5 c
Metabolic acidosis decreases the apicalCl /HCO 3 − exchanger activity in rabbit -ICs ( 25, 30, 32, 33, 40 ). However, little is knownabout the regulation of the apicalCl /HCO 3 − exchanger in -ICs of either control or acidotic rats. Furthermore, no study has examined the molecular adaptation of -IC apicalCl /HCO 3 − exchanger in acid-basedisorders. In the present study, we sought to correlate the kidneyexpression of pendrin with the apicalCl /HCO 3 − exchanger activity in single -ICs in rats subjected to acid loading.

METHODS6 Q1 L5 s) ^3 S' r' c
& d, K& j( `9 Z# `
Animals. Metabolic acidosis was generated according to established protocols( 1, 9 ). Female Sprague-Dawley rats, 100-150 g, were given 280 mM NH 4 Cl in their drinking water for 4 days.Serum HCO 3 − was 14 ± 1.2 mM in rats onNH 4 Cl, consistent with metabolic acidosis vs. 24 ± 1.5 mM in control ( P n = 4 foreach). Both groups were allowed free access to water and food.: h. s' G0 Q/ ^, K, e
5 d9 [4 y8 g! R' V0 ~9 F
Isolation of CCDs and in vitro microperfusion. Rats were killed by intraperitoneal injection of pentobarbital sodium(100 mg/kg of body wt). Kidneys were quickly removed and placed inice-cold dissection medium ( solution 1; Table 1 ). Thin coronal slices (~1 mm) werecut and transferred to the dissection chamber. CCDs were obtained byfreehand dissection. Dissected tubules were quickly transferred to the1.5-ml temperature-controlled specimen chamber mounted on an invertedZeiss Axiovert S-100 microscope (Carl Zeiss, Thornwood, NY). Tubuleswere perfused by using concentric glass pipettes according to themethod of Burg and colleagues ( 7, 8 ) with modifications( 40 ) at 5-cm water pressure. Solutions used to perfuse andbath the tubules are listed in Table 1. Solutions were delivered to thespecimen chamber in tubing impermeable to CO 2 andO 2 (Cole Palmer, Chicago, IL) by a peristaltic pump(Peristar, WPI, Sarasota, FL) at a rate of 1 ml/min. Fluid in thechamber was constantly superfused with 95% O 2 -5%CO 2 to minimize gas loss and help keep the pH of the bathfluid constant. Chamber pH was frequently checked on a pH meter (modelB213, Horiba). Initially, tubules were perfused with fast green dye(Sigma, St. Louis, MO) to identify the damaged cells, because damagedcells take up the dye. Tubules were carefully inspected and discarded if damaged cells were found ( 41 ).; p9 c' G5 I" n4 M% p

Table 1. Solutions utilized in microperfusion experiments- p, E0 r9 X1 \( W: ~2 p

Intracellular pH measurement in intercalated cells. After 15-20 min equilibration in solution 2, the tubulewas perfused with 5 µM2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein AM (BCPCF-AM)for 5 min. Only intercalated, but not principal, cells take up thepH-sensitive dye when perfused from the luminal side ( 45 ).BCPCF-AM is a close analog of BCECF-AM, with improved spectralcharacteristics (a higher absorption at isosbestic point yields abetter signal-to-noise ratio) ( 16 ). In our preliminary experiments, we noticed that BCPCF-AM was better retained in rat intercalated cells. Fluorescent measurements were done with the ZeissAxiovert S-100 inverted microscope equipped with Attofluor RatioVisiondigital imaging system (Attofluor, Rockville, MD). An Achroplan×40/0.8 water objective with 3.6-mm working distance was used.Excitation wavelengths were recorded at 488 and 440 nm, and emissionwas measured at 520 nm. Attofluor RatioVision software allowed for"regions of interest" to be applied to individual cells so thatmultiple cells in a single tubule were simultaneously examined.Generally, three to seven cells were examined per tubule. Only onetubule per animal was examined. Digitized images were analyzed by usingAttograph software (Attofluor). Intracellular calibration wasperformed by using the high-K -nigericin method ( 24, 30, 33, 37, 45 ). A three-point calibration curve was used toconvert the recorded ratios into intracellular pH (pH i )values. pH clamp calibration values (7.5, 7.0, and 6.5) were recordedfrom each cell that was selected for experimental measurements in everytubule at the end of the experiment.
0 Q' B- [. J: P  A  F1 L
Apical Cl /HCO 3 − exchanger activity in -ICs was assessed as the rate of pH i change (calculatedas a linear tangent to the initial pH change) as well as amplitude ofpH i response when the luminal perfusate ( solution2; Table 1 ) was switched to a Cl -free solution( solution 3; Table 1 ). This maneuver causes cell alkalinization in -ICs via reversal of the apicalCl /HCO 3 − exchanger, whereaspH i of -ICs remains unchanged. When pH i stabilization occurred in Cl -free medium, the luminalperfusate was switched back to Cl -containing solution,resulting in recovery of pH i to baseline levels viaCl /HCO 3 − exchange ( 24, 30, 32, 33, 45, 46 ).

In separate maneuvers, bath solution was switched fromCl -containing ( solution 2; Table 1 ) to aCl -free solution ( solution 3; Table 1 ). Underthis protocol, -ICs alkalinize whereas -ICs acidify ( 24, 30, 32, 33, 45, 46 ). The alkalinization of -ICs in responseto the removal of bath Cl is due to the reversal ofbasolateral Cl /HCO 3 − exchanger. Theacidification of -ICs in response to bath Cl removalis due to the stimulation of apicalCl /HCO 3 − exchanger, as intracellularCl exits the cell via basolateral Cl conductance. This increases the inward gradient for luminalCl and therefore stimulates the apicalCl /HCO 3 − exchanger ( 46 ).* d- N# R. k9 [
6 I) w- p1 |/ a  R- Q1 q
RNA isolation and Northern blot hybridization. Total cellular RNA was extracted from cortex by the method ofChomczynski and Sacchi ( 10 ), quantitatedspectrophotometrically, and stored at 80°C. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel andtransferred to Magna NT nylon membranes (MSI). Membranes werecross-linked by ultraviolet light and baked for 1 h. Hybridizationwas performed according to Church and Gilbert ( 11 ). Apendrin-specific cDNA probe ( 36 ) was labeled with[ 32 P]deoxynucleotides by using the Rad-Prime DNA labelingkit (GIBCO-BRL). The membranes were washed, blotted dry, exposed to aPhosphorImager cassette at room temperature for 24-72 h, and readby the PhosphorImager (Molecular Dynamics, Sunnyvale, CA).% a0 W5 ?0 z0 n" m( Z! T
2 h. F/ w% C& X& o: k
For pendrin hybridization, a PCR fragment encoding nucleotides1473-1961 was generated from rat kidney using oligonucleotide primers 5'-CAT TCT GGG GCT GGA CCT C and 5'-CCT TCG GGA CAT TCA CTT TCAC that were designed on the basis of rat pendrin cDNA (GenBankaccession no. AF-167412).9 P9 \, X2 V* Y* ^9 R

Nephron segment RT-PCR. CCDs were dissected as single-nephron segments from freshly killednormal or acidotic rat kidneys at 4-6°C as described( 36 ). The dissection media comprises 140 mM NaCl, 2.5 mMK 2 HPO 4, 2 mM CaCl 2, 1.2 mMMgSO 4, 5.5 mM D -glucose, 1 mM Na citrate, 4 mMNa lactate, and 6 mM L -alanine, pH 7.4, and bubbled with100% O 2. Tubule lengths were ~0.5-0.7 mm for bothcontrol and acidotic animals. For each RT-PCR, two nephron segments(CCDs) from each rat were pooled in a small volume (5-10 µl) ofice-cold PBS. The tubules were centrifuged at 12,000 g for 1 min at room temperature, and the PBS was removed and replaced with 10 µl of a tubule lysis solution consisting of 0.9% Triton X-100, 5 mMDTT, and 1 U/µl rRNasin (Promega). After 5 min on ice, the tubuleswere gently agitated by tapping the tube, and 1 µl (0.5 µg)oligo(dT) primer, 1 µl H 2 O, 4 µl 5× reversetranscription buffer, 2 µl DTT (0.1 M), and 1 µl dNTPs (10 mM each)were added. The reaction was equilibrated to 42°C for 2 min, and 1 µl SuperScript II RT (Life Technologies) was added, mixed, andincubated for 1 h at 42°C. After reverse transcription, 30 µlof TE (10 mM Tris · Cl and 1 mM EDTA, pH 8.0)were added, and the combined mixture was heated to 95°C for 5 min andplaced on ice.
, D% n1 f9 ^' z% I+ D6 t8 g: ~
The following oligonucleotide primers (5'-CAT TCT GGG GCT GGA CCT C and5'-CCT TCG GGA CAT TCA CTT TCA C) were designed on the basis of ratpendrin cDNA (GenBank accession no. AF-167412) and used for nephronsegment RT-PCR. These primers should amplify a PCR fragment of488 bp. Amplification of the pendrin cDNA by the PCR was performed byusing parameters previously established with rat CCD ( 36 ).Briefly, each PCR contained 10 µl cDNA, 5 µl 10× PCR buffer (with20 mM MgCl 2 ), 1 µl 10 mM dNTPs, 10 pmol/primer, and 2.5 µl Taq DNA polymerase in a final volume of 50 µl.Cycling parameters were 95°C for 45 s, 47°C for 45 s, and72°C for 2 min. The expression of -actin was examined in eachsample, and pendrin/ -actin mRNA ratios were calculated in controland acidosis and compared. Four separate CCD segment samples from twocontrol and two acidotic rats (2 samples/rat) were isolated and examined.
, ^2 g3 c; x9 c) E
Immunocytochemistry: antibodies. For pendrin, polyclonal antibodies were raised in two rabbits against asynthetic peptide corresponding to amino acids 734-752 (CKSREGQDSLLETVARIRDC). The sequence of the synthetic peptide used forantibody generation was identical for rat, mouse, and human pendrin.Antibodies were purified with cystein-affinity columns. This antibodyis highly specific and labels the apical membranes of a subset of CCDcells ( 35 ).* w3 [* d* C6 ^1 w0 w$ r( J& v

For aquaporin 2 (AQP2), peptide-derived polyclonal antibodies specificto the AQP2 water channel were raised in our laboratory as described( 2 ). The rat AQP2 peptide has the followingsequence: NH 2 -CEVRRRQSVELHSPQSLPRG- SKA-COOH, whichcorresponds to amino acid residues 250-271 of the COOH-terminaltail of the vasopressin-regulated AQP2 water channel. This antibody ishighly specific and has been successfully used to examine theregulation of AQP2 in pathophysiological disorders ( 2 ).

Immunofluorescense. Animals were killed with an overdose of pentobarbital sodium andperfused through the left ventricle of the heart with 200 ml of 0.9%saline followed by cold 500 ml of 4% paraformaldehyde in 0.1 Msodium-phosphate buffer (pH 7.4). Kidneys were removed, cut in tissueblocks, and left in the same fixative solution overnight at 4°C.6 r5 Z. O, s0 `6 j  o
" k0 C% K6 S8 p! Y" A$ T
For cryosections, tissue blocks were removed from the fixative solutionand soaked in 30% sucrose overnight. The tissue was frozen on dry ice,and 5-µm sections were cut with a cryostat and stored at 80°Cuntil use. For staining, cryosections were washed twice in 0.01 M PBS(pH 7.4) and blocked with 10% goat serum-0.3% Triton X-100-PBSsolution for 45-60 min. Primary pendrin antibody was diluted 1:40in 1% BSA-0.3% Triton X-100-PBS solution and applied to sectionsovernight at room temperature. Primary AQP2 antibody was diluted 1:20in 1% BSA-0.3% Triton X-100-PBS solution and applied to sectionsovernight at room temperature. Sections treated with either primaryantibody were rinsed twice in 0.01 M PBS for 10 min and then incubatedwith a secondary antibody for 2 h at room temperature. RhodamineTRITC (Jackson Immunoresearch Laboratories, West Grove, PA)-conjugatedgoat-anti-rabbit IgGs was used as secondary antibody for pendrin (1:200dilution). In some sections we used green fluorescent secondary, Oregongreen-conjugated goat-anti-rabbit IgGs (Molecular Probes, Eugene, OR)at a dilution of 1:150, because we noticed that there was lessbackground with the green fluorescent secondary dye. Sections were thenwashed four times, air dried, and mounted in Vectashield mountingmedium for fluorescence (Vector Laboratories, Burlingame, CA). Sections were examined, and images were acquired on the Nikon PCM 2000 laserconfocal scanning microscope as 0.5-1 µm "optical sections" of the stained cell membrane; a ×20 objective and ×60 oil-immersion objective were used. The 543.5-nm single-line output of the HeNe laserwas used for the red dye excitation, and the standard red channellong-pass 565-nm filter was used as an emission filter. A standardargon laser 488-nm line and 515/30-nm emission filter were used for thegreen- emitting dye. Instrument settings, black level, gain, andintegration time, which is pixel dwell time, were kept the same whensections from control and acid-loaded animals were compared. Inaddition, all sections from control and acidotic animals were processedthe same day and with the same dilutions of primary and secondaryantibodies. More than 20 sections from four separate animals wereexamined in each group of control or acidosis.

Materials. [ 32 P]dCTP was purchased from New England Nuclear (Boston,MA). The RadPrime DNA labeling kit was purchased from GIBCO-BRL.BCECF-AM and BCPCF-AM were from Molecular Probes. Nitrocellulosefilters and and all other chemicals were purchased from Sigma.Nigericin was dissolved in ethanol as 10 mM stock and diluted 1:1,000for the final concentration of 10 µM.

Statistics. Results are expressed as means ± SE. Statistical significancebetween experimental groups was determined by Student's t -test, as required. Significance was asserted if P of the number of intercalatedcell types in control and acidotic animals was analyzed by 2 -test as computed on SAS software (version 8, SASInstitute, Cary, NY).
2 u) }7 U$ }2 P/ w- E
RESULTS- Y8 J9 M$ i4 B: `

mRNA expression of pendrin in the kidneys of control and acidoticrats. In the next series of experiments, we sought to examine the effect ofmetabolic acidosis on the expression of pendrin in rat kidney. Ratswere made acidotic by addition of NH 4 Cl to their drinkingwater (see METHODS ). Animals were killed, and kidney RNAand sections were utilized for expression studies. Figure 1 A is arepresentative Northern blot hybridization experiment and demonstratesthat the mRNA expression of pendrin in the kidney cortex is decreasedin metabolic acidosis. The results of four samples fromseparate animals, (summarized in Fig. 1 B ) indicate that theexpression of pendrin is decreased by 68% in acidotic rats( n = 4, P

Fig. 1. A : expression of pendrin mRNA (PDS) in thekidneys of control and acidotic rats. Representative Northern blothybridizations indicate the downregulation of pendrin mRNA in kidneycortices of acidotic rats. The results of 4 separate samples from 4 separate animals indicate that the expression of pendrin is decreasedby 68% in acidotic rats ( P B : summary of results. PendrinmRNA/28S rRNA ratios ( n = 4 for each bar). C : nephron segment RT-PCR with primers specific for pendrin.A representative ethidium bromide staining of agarose gel( left and right ) demonstrates a PCR product ofexpected size (488 bp) for rat pendrin in the cortical collecting duct(CCD) of control and acidotic rats. The expression of -actin mRNA isshown as control in the same nephron segments. No PCR product wasobserved in the negative (no RT) reactions (not shown). For statisticalanalysis, pendrin/ -actin mRNA ratios from 4 different CCD samplesfrom normal and acidotic rats (2 animals/each group) were acquired andanalyzed by ANOVA. The expression of pendrin decreased by 62% inkidneys of rats with acidosis ( P) F1 ?- ]% h1 T

Pendrin mRNA is detected in both proximal tubule and CCD in normalkidney ( 36 ). To assess the CCD-specific regulation, we examined the mRNA expression of pendrin in acidosis by nephron segmentRT-PCR. Single CCDs were isolated from kidneys of control andacid-loaded rats as before ( 36 ) and subjected to RT-PCR. Figure 1 C shows a representative ethidium bromide gel imagefrom semiquantitative nephron segment RT-PCR experiments anddemonstrates that pendrin mRNA is decreased in acidosis. -Actin mRNAexpression was measured in the same samples and is shown as control.The expression of pendrin, as assessed by semiquantitativependrin/ -actin mRNA ratio, decreased by 62% in CCDs of acidoticrats ( P n = 4, separate CCDsamples from 2 separate rats/each group). These results indicate thatthe reduction in the cortical expression of pendrin in acidosis (Fig. 1 A ) is in part due to downregulation in CCD.

Immunofluorescent staining of pendrin in the kidneys of control andacidotic rats. In the next series of experiments, the effect of acidosis on pendrinabundance was examined by indirect immunofluorescence. In kidneysections from control rats, immunofluorescent staining with thepurified polyclonal pendrin antibody (Fig. 2 A ) shows apical labeling in asubpopulation of CCD cells. Specificity of the staining is demonstratedin Fig. 2 B. As indicated, labeling was completely preventedby preadsorption of the immune sera with the synthetic peptide. Thelimited expression of pendrin in CCD is in agreement with recentreports on the expression of pendrin in the kidney ( 28, 35 ).0 v: r3 k$ k$ l0 p) i& d5 [
1 ]; S* c* P. I, d! h6 k, f4 F0 j
Fig. 2. Specificity of pendrin immunofluorescent labeling. A : immunocytochemical staining of rat kidney with pendrinpolyclonal purified antibody. As indicated, the antibody labeled theapical membranes in a subpopulation of CCD cells. B :labeling was completely prevented by preadsorption of the antibody withthe synthetic peptide.
! p  d5 H5 a5 l3 I
As shown at a higher magnification, the apical staining in CCDsdecreased in acidotic rats vs. control animals (Fig. 3 A, bottom and top,respectively). Because of a high background with the secondaryred fluorescent dye, we repeated the experiments with a greenfluorescent dye to tag the pendrin antibody. Figure 3 B represents an experiment that was performed with a green fluorescent dye and shows decreased intensity of pendrin immunostaining in kidneysof rats with acidosis vs. control animals (Fig. 3 B, bottom and top, respectively). A broader viewcompares pendrin expression in control and acidotic animals (Fig. 3 C, top and bottom, respectively).1 x( D& D( K0 Q& J2 T. ~5 J

Fig. 3. Pendrin expression in metabolic acidosis. A :pendrin immunofluorescent staining with the use of a red fluorescentsecondary dye. As indicated, the apical labeling with pendrin antibodyin CCDs decreased in acidotic rats ( bottom ) compared withcontrol animals ( top ). PT, proximal tubule. B :pendrin immunofluorescent staining with the use of a green fluorescentsecondary dye. As indicated, the apical labeling with pendrin antibodyin CCDs decreased in acidotic rats ( bottom ) compared withcontrol animals ( top ). C : pendrinimmunofluorescent staining (low magnification) of kidney sections incontrol ( top ) and acidotic animals ( bottom ). Whenimages from the kidney sections of control and acid-loaded rats werecompared, instrument settings (camera gain, black level, andintegration time) were kept the same at both magnifications. Allsections were fixed and stained the same day and examined with the sameconcentration of primary and secondary antibodies. Arrows, typicalapical staining pattern of pendrin in a subpopulation of CCD cells.Magnification, ×600.) D( {- \! x, T* j
/ v3 ^1 ?3 V" X
In the next series of experiments, we examined the expression of anunrelated transporter in CCD in rats subjected to NH 4 Cl loading to determine the specificity of pendrin regulation in acidosis.Accordingly, the expression of AQP2 was examined by immunofluorescentlabeling in rats with acidosis using AQP2-specific antibodies raised inour laboratory ( 2 ). Consistent with published reports,AQP2 staining (Fig. 4 A ) isobserved on the apical membrane of the majority of CCD cells( 23 ). Contrary to pendrin, AQP2 labeling does not decreasein kidneys of acidotic animals (Fig. 4 B ). Taken together,these experiments indicate that metabolic acidosis decreases theexpression of pendrin in rat CCD.

Fig. 4. Immunofluorescent staining of AQP2. A :representative immunofluorescent staining of kidney AQP2 in normalrats. B : representative immunofluorescent staining of kidneyAQP2 in acidotic rats. Results demonstrate that AQP2 labeling is notdecreased in acidosis. Sections were made from the same animals thatwere used for pendrin immunolabeling. Magnification,×600.( u. @' h% ^1 d
) C* g5 R9 |5 w9 h+ M& L
Cl /HCO 3 − exchanger activity in intercalated cells of control and acidoticanimals. The results of the above studies demonstrated decreased expression ofpendrin in CCD in metabolic acidosis. To correlate these results withfunctional studies, the apical Cl /HCO 3 − exchanger activity in -ICs was examined in control and acidoticanimals. Toward this end, the -ICs and -ICs were first identifiedby their pH i response to luminal or basolateralCl removal in microperfused CCDs, according to theestablished criteria (see METHODS ). RepresentativepH i tracings in -ICs in control and acidosis are shownin Fig. 5, A and B,respectively.

Fig. 5. Apical Cl /HCO 3 − exchanger activityin -intercalated cells ( -IC) of control and acidotic animals. A : representative intracellular pH (pH i ) tracingof an individual normal -IC demonstrating cell alkalinization whenluminal Cl was removed and cell acidification when bathCl was removed. B : representativepH i tracing of an individual -IC from an acidotic rat. C : rate of intracellular alkalinization when luminalCl was removed in -ICs in normal and acidotic rats. Asshown, apical Cl /HCO 3 − exchangeractivity is significantly decreased in -ICs in acidosis. D : amplitude of pH i alkalinization when luminalCl was removed in -ICs in normal and acidotic rats. Asshown, the magnitude of intracellular alkalinization in response toluminal Cl removal is significantly diminished in -ICsin acidosis. E : rate of intracellular acidification whenbath Cl was removed in -ICs in normal and acidoticrats is significantly decreased in acidosis. F : amplitude ofpH i acidification when bath Cl was removed in -ICs in normal and acidotic rats. As shown, the magnitude ofintracellular acidification in response to bath Cl removal is significantly diminished in -ICs in acidosis.3 Y$ O; O" K3 J9 \3 O" y$ ~

In the control group, 15 of 44 cells that were labeled with BCPCF-AM in10 CCDs alkalinized when luminal Cl was removed at a rateof 0.16 ± 0.02 pH units/min (Fig. 5 C ). The cell pHincreased from a baseline of 7.21 ± 0.01 to 7.39 ± 0.02 inresponse to luminal Cl removal, with a change in pH( pH) of 0.18 ± 0.02 (Fig. 5 D ). The same cellsacidified when basolateral Cl was removed at a rate of0.12 ± 0.02 pH units/min (Fig. 5 E ). The cell pHdecreased from a baseline of 7.20 ± 0.02 to 7.03 ± 0.03 inresponse to bath Cl removal, with a pH i of0.16 ± 0.02 (Fig. 5 F ). These cells were thereforeconsidered -ICs. There seemed to be fewer -ICs in CCDs fromacidotic animals (9 -ICs of 41 intercalated cells) compared withcontrol CCDs (15 -ICs of 45 intercalated cells). However, thedifference did not reach statistical significance as assessed by 2 -test ( P = 0.24).+ u' S& t) V7 f

In acidotic rats, 9 of 41 cells that were labeled with BCPCF-AM in nineCCDs alkalinized when luminal Cl was removed (and hencewere identified as -ICs) at a rate of 0.10 ± 0.01 pH units/min(vs. 0.16 ± 0.02 in normal rats, P 0.05, n = 9) (Fig. 5 C ). The cell pH increased froma baseline of 7.26 ± 0.01 to 7.32 ± 0.02 in response toluminal Cl removal, with a pH i of0.06 ± 0.02 ( P incontrol animals) (Fig. 5 D ). Baseline pH was notstatistically different from control animals (7.26 ± 0.01 inacidotic vs. 7.21 ± 0.01 in control animals, P 0.05). The rate of acidification when basolateral Cl wasremoved was 0.04 ± 0.003 pH units/min ( P ± 0.02 in control animals) (Fig. 5 E ). Thecell pH decreased from 7.26 ± 0.04 to 7.17 ± 0.04, with a pH i of 0.07 ± 0.02 ( P vs.0.16 ± 0.02 in control) (Fig. 5 F ).

In control animals, 24 of 44 cells that were labeled with BCPCF-AM in10 CCDs alkalinized when basolateral Cl was removed at arate of 0.22 ± 0.02 pH units/min (Fig. 6 C ). These cells weretherefore considered -ICs. Representative pH i tracingsin -ICs in control and acidosis are shown in Fig. 6, A and B, respectively. The cell pH increased from a baselineof 7.22 ± 0.04 to 7.43 ± 0.02 in response to basolateralCl removal, with a pH of 0.21 ± 0.02 (Fig. 6 D ). Luminal Cl removal did not significantlychange pH i of these cells (from a baseline pH i of 7.29 ± 0.01 to 7.28 ± 0.02, P 0.05).
5 c' X/ L! t2 i- S: L7 Q% W/ s
Fig. 6. Basolateral Cl /HCO 3 − exchangeractivity in -intercalated cells ( -ICs) of control andacidotic animals. A : representative pH i tracingof an individual normal -IC demonstrating no pH i changeswhen luminal Cl was removed but cell alkalinization whenbath Cl was removed. B : representativepH i tracing of an individual -IC from an acidotic rat. C : rate of intracellular alkalinization when bathCl was removed in -ICs in normal and acidotic rats. Asshown, basolateral Cl /HCO 3 − exchangeractivity is significantly increased in -ICs in acidosis. D : amplitude of pH i alkalinization when bathCl was removed in -ICs in normal and acidotic rats. Asshown, the magnitude of intracellular alkalinization in response tobath Cl removal is significantly increased in -ICs inacidosis.6 Q7 M$ P7 x' ^8 ~: y$ v3 {

In acidotic rats, 29 of 41 cells that were labeled with BCPCF innine CCDs alkalinized when basolateral Cl was removed(and hence were designated as -ICs) at a rate of 0.28 ± 0.02 pH units/min ( P animals) (Fig. 6 C ). Cell pH increased from abaseline of 7.29 ± 0.01 to 7.53 ± 0.01 in response to bathCl removal, with a pH of 0.24 ± 0.01 ( P in control) (Fig. 6 D ). Luminal Cl removal did not change thecell pH of -ICs (from a baseline of 7.29 ± 0.01 to 7.3 ± 0.02, P 0.05). In addition to -ICs and -ICsthat express Cl /HCO 3 − exchanger on theirbasolateral and luminal membranes, respectively, a subtype ofintercalated cells in rabbit kidney has been described byseveral investigators that expressesCl /HCO 3 − exchanger activity on itsapical and basolateral membrane ( 13, 47 ). We observed that5 of 44 cells in control animals alkalinized when either luminal orbasolateral Cl was removed at the rate of 0.17 ± 0.03 or 0.22 ± 0.04 pH units/min, respectively. In acidoticanimals, 3 of 41 cells alkalinized when either luminal or basolateralCl was removed at the rate of 0.19 ± 0.031 or0.22 ± 0.04 pH units/min, respectively. Although there was nosignificant difference between the rate of apical or basolateralCl /HCO 3 − exchanger in these cells inacidosis vs. control ( P 0.05), no firm conclusioncould be reached at this stage due to the low abundance of these cells.A representative pH i tracing of this cell type is shown inFig. 7.# |- g& D# \% i% V7 w
# B/ U. `4 n1 w
Fig. 7. A representative pH i tracing of an individualintercalated cell demonstrating cell alkalinization when either luminalor bath Cl was removed." C# t, c' y* j4 X. t

DISCUSSION/ N4 m/ T8 K6 U% U0 S* T* c3 K

The present experiments examine the regulation of pendrinin metabolic acidosis in rat kidney. Functional studies inmicroperfused cortical collecting tubules demonstrate that apicalCl /HCO 3 − exchanger activity in -ICsis decreased by ~60% in acid-loaded rats (Fig. 5 ). Northern blot hybridization experiments demonstrated that the mRNA expression ofpendrin decreased in the kidneys of acidotic animals (Fig. 1 ).Similarly, immunohistochemical studies indicated decreased expressionof pendrin protein in intercalated cells of acid-loaded rats (Figs. 2 and 3 ). In contrast to the apicalCl /HCO 3 − exchanger in -ICs, thebasolateral Cl /HCO 3 − exchanger activityin -ICs increased in acidosis (Fig. 6 ).
% I8 O- u8 M: h% b  Y! }4 A
A Cl /HCO 3 − exchanger is located on theapical membrane of -ICs and mediates the secretion ofHCO 3 − into the lumen of CCD. Contrary to -ICs, -ICs express a Cl /HCO 3 − exchanger ontheir basolateral membrane, which mediates the reabsorption ofHCO 3 − in CCDs ( 31 ). This transporter is atruncated splice variant of red cell AE1 or band 3 ( 6 ). Onthe basis of immunohistochemical studies indicating a lack of AE1staining on the apical membranes of CCD cells, as well as functionalstudies showing differences in Cl affinity and DIDS sensitivity, it wasconcluded that the Cl /HCO 3 − exchanger of -ICs is distinct from AE1 ( 31 ).
6 ?8 D$ Z7 r( R6 w' S# F
Recent findings identified pendrin as an important candidate foran apical Cl /HCO 3 − exchanger in -ICs( 36 ). This conclusion was on the basis of functional andmolecular studies indicating that pendrin is an apicalCl /HCO 3 − exchanger in the kidney cortex, with abundant expression in rat CCD ( 36 ). This wassupported by immunocytochemical studies demonstrating apicallocalization of pendrin in a subset of CCD cells in mouse, rat, andhuman kidney that were distinct from -ICs and principal cells( 28 ). In addition, pendrin null mice failed to secreteHCO 3 − in their CCDs when subjected toHCO 3 − loading, indicating the important role ofpendrin in adaptation of the mouse CCD to change in alkali load( 28 ). These findings are consistent with pendrinfunctioning as an apical Cl /HCO 3 − exchanger and mediating HCO 3 − secretion into rat andmouse CCD. CCDs from control or acid-loaded rats absorbHCO 3 − ( 4, 15 ). Immunocytochemical data inrat kidney show that adaptation to systemic acidosis results fromdecreased -ICs function, as concluded from changes inH -ATPase- and AE1-labeling patterns. The present studiesare the first to measure the apicalCl /HCO 3 − exchanger activity in -ICsin rat kidney (Fig. 5 ). Furthermore, our findings demonstratingdecreased apical Cl /HCO 3 − exchangeractivity in rat -ICs (Fig. 5 ) are in agreement with previousimmunocytochemical data indicating adaptive regulation ofH -ATPase in rat kidney in acidosis ( 5, 29, 38 ). The reduction in pendrin mRNA in acidosis is specific, asjudged by a lack of reduction in AQP-2 mRNA in acidosis( 3 ). These latter results correlate very well withimmunocytochemical labeling performed in the present studies (Fig. 4 ).The reduction in apical Cl /HCO 3 − exchanger activity correlates with a significant downregulation inpendrin expression (Figs. 1 and 2 ).) s6 a! [0 Y# H6 W7 _; t/ ?) s" |

Rat kidney AE1 mRNA levels increased in response to both respiratoryand metabolic acidosis (11a, 18). Immunocytochemical studies indicatedadaptive upregulation of AE1 in basolateral membranes of -ICs inacidotic rats ( 21, 29, 38, 43 ). Our results demonstratingincreased basolateral Cl /HCO 3 − exchangeractivity in -ICs in acidosis are in complete agreement with theabove molecular and immunocytochemical studies.4 c4 L+ l: ]1 k  ]2 l9 X2 W

Adaptation of rabbit CCD to both in vivo and in vitro metabolicacidosis has been studied in detail and shown to result mainly from thedecreased activity of the apicalCl /HCO 3 − exchanger in -ICs, as wellas the increased activity of the basolateralCl /HCO 3 − exchanger of -ICs andreversal of the functional polarity of -ICs in CCDs incubated in lowpH in vitro ( 26, 30, 32, 33 ). However, molecular identity of the Cl /HCO 3 − exchanger mediatingthese changes is presently unknown. Tsuganezawa et al.( 39 ) have recently cloned the apicalCl /HCO 3 − exchanger of rabbit -ICs and named it AE4. AE4 mRNA expression has also been shown in rat kidney ( 26 ), but preliminary immunocytochemical labeling fromdifferent groups has been conflicting with respect to its subcelullarlocalization in rat CCDs ( 12, 26 ). Further studies arenecessary to address this issue.

Some studies in rabbit CCDs have shown both apical and basolateralCl /HCO 3 − exchanger activity inintercalated cells ( 13, 47 ). In addition,immunocytochemical studies have identified a group of intercalatedcells referred to as non-A-non-B intercalated cells in rat and mouseCCDs ( 21, 38, 43 ). Interestingly, we have observed a smallnumber of cells that have apical as well as basolateralCl /HCO 3 − exchanger activity (Fig. 7 ), which may resemble non-A-non-B intercalated cells identified by immunocytochemical staining. However, because of the low abundance ofthese cells, it is hard to draw any firm conclusions with regard totheir adaptation in acidosis., E* C3 B; r8 ~1 _6 f9 ~

In conclusion, mRNA expression and protein abundance of pendrin aredownregulated in metabolic acidosis in the rat kidney, resulting indecreased apical Cl /HCO 3 − exchangeractivity in -ICs. Taken together, these results suggest that pendrinplays an important role in HCO 3 − secretion and, as aresult, in acid-base regulation in rat kidney.

ACKNOWLEDGEMENTS
9 W+ X" j# I  l+ J' Y0 U8 H1 i* q/ V
These studies were supported by a Merit Review grant from theDepartment of Veterans Affairs, National Institute of Diabetes andDigestive and Kidney Diseases Grants DK-52821 and DK-54430, a CysticFibrosis Foundation grant, and grants from Dialysis Clinic, Inc.
      【参考文献】
1. Amlal, H,Chen Q,Greeley T,Pavelic L,andSoleimani M. Coordinated down-regulation of NBC-1 and NHE-3 in sodium and bicarbonate loading. Kidney Int 60:1824-1836,2001  .* y$ r1 N" b1 ?- }
2 D$ `8 q) P  B, D' E, p9 `* g9 [
: D0 S3 `' _: J
& j4 q/ [7 f( E; x7 p
2. Amlal, H,Chen Q,Habo K,Wang Z,andSoleimani M. Fasting downregulates renal water channel AQP2 and causes polyuria. Am J Physiol Renal Physiol 280:F513-F523,2001 .* P- M8 R' A- s/ q9 y6 D3 @

3 J/ ?) P7 @  l  f) n% F

3. Amlal, H,Chen Q,andSoleimani M. Metabolic acidosis upregulates the expression of aquaporin-2 (AQP-2) water channel in rat kidney (Abstract). J Am Soc Nephrol 11:A0067,2000.5 D" m: k" k3 |, k2 [$ w- l$ W2 F
. m" L0 s: ~. \! e8 J# V1 [- b2 e

4. Atkins, JL,andBurg MB. Bicarbonate transport by isolated perfused rat collecting ducts. Am J Physiol Renal Fluid Electrolyte Physiol 249:F485-F489,1985 .% t  J' C" W1 ~

2 c5 I1 U' N* |: T7 p& B& G; |

5. Bastani, B,Purcell H,Hemken P,Trigg D,andGluck S. Expression and distribution of renal vacuolar proton-translocating adenosine triphosphatase in response to chronic acid and alkali loads in the rat. J Clin Invest 88:126-136,1991  .* h0 o1 l, F% ^
( J3 I6 S8 a- V( r' C
. q. y: ~# w% u) W, l% B
" W" l' H, D. H/ c) V, `, ~/ P) T
6. Brosius, FC, 3rd,Alper SL,Garcia AM,andLodish HF. The major kidney band 3 gene transcript predicts an amino-terminal truncated band 3 polypeptide. J Biol Chem 264:7784-7787,1989 .
! m5 N+ ?. G9 \% k/ Q6 O% D

7. Burg, M,Grantham J,Abramow M,andOrloff J. Preparation and study of fragments of single rabbit nephrons. Am J Physiol 210:1293-1298,1966 .) J/ {9 Z8 N2 E, H
0 m+ @' M  x* a) a1 j, ?$ \
  U1 D# ?0 {* a) Z7 I! X

8. Burg, M,andGreen N. Bicarbonate transport by isolated perfused rabbit proximal convoluted tubules. Am J Physiol Renal Fluid Electrolyte Physiol 233:F307-F314,1977 .5 L9 m# J. l: k" D. w' t' y' h- B: V
% w! d, L# z5 G# m4 o
" v+ L. s5 z/ A" c7 ^6 @6 ?* ~/ k

9. Burnham, CE,Flagella M,Wang Z,Amlal H,Shull GE,andSoleimani M. Cloning, renal distribution, and regulation of the rat Na -HCO 3 − cotransporter. Am J Physiol Renal Physiol 274:F1119-F1126,1998 .  B. y/ m* D: p# R& v

( s; c0 j$ Z0 N4 J: I+ j
+ V  E- w3 z5 s
10. Chomczynski, P,andSacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159,1987  .  q6 T# d2 w: K0 Y
' ]2 p$ c- ^# T3 @6 |5 W
3 W! |. l" g0 b9 k' Y
( n: R- v# e2 e; z* F6 r
11. Church, GM,andGilbert W. Genomic sequencing. Proc Natl Acad Sci USA 81:1991-1995,1984 .. h5 w. O; a6 \# ^6 V5 h3 ~* ^; R

8 Q9 @6 l& v) ~( b' g

11a. Da Silva, JC, Jr,Perrone RD,Johns CA,andMadias NE. Rat kidney band 3 mRNA modulation in chronic respiratory acidosis. Am J Physiol Renal Fluid Electrolyte Physiol 260:F204-F209,1991 .
. \) B1 g: V9 n! R9 ]
: X2 ]- b+ L9 s# v/ R- c
7 F: b5 J5 P2 p1 x' ^6 B" N) y# G
12. Elkjaer, ML,Hager H,Ishibashi K,Muallem S,Frokier J,andNielsen S. Laser confocal microscopical and immunoelectron microscopical localization of anion exchanger AE4 in rat kidney (Abstract). J Am Soc Nephrol 12:A0013,2001.

" h/ E. k; [; H( x
13. Emmons, C,andKurtz I. Functional characterization of three intercalated cell subtypes in the rabbit outer cortical collecting duct. J Clin Invest 93:417-423,1994  .
8 a/ Z9 L1 U3 N5 s' A

14. Everett, LA,Glaser B,Beck JC,Idol JR,Buchs A,Heyman M,Adawi F,Hazani E,Nassir E,Baxevanis AD,Sheffield VC,andGreen ED. Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat Genet 17:411-422,1997  .5 o3 p/ i9 P- Y& |6 _

15. Gifford, JD,Sharkins K,Work J,Luke RG,andGalla JH. Total CO 2 transport in rat cortical collecting duct in chloride-depletion alkalosis. Am J Physiol Renal Fluid Electrolyte Physiol 258:F848-F853,1990 .2 w) M4 c: x9 y8 N- l

16. Haugland RP. pH indicators. In: Handbook of FluorescentProbes and Research Products (Chapter 21) . http://www.probes.com/handbook/sections/2100.html [2001].

17. Hoglund, P,Haila S,Socha J,Tomaszewski L,Saarialho-Kere U,Karjalainen-Lindsberg ML,Airola K,Holmberg C,De la Chapelle A,andKere J. Mutations of the Down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea. Nat Genet 14:316-319,1996  ./ b$ ]1 ~5 H  y# X

18. Huber, S,Asan E,Jons T,Kerscher C,Puschel B,andDrenckhahn D. Expression of rat kidney anion exchanger 1 in type A intercalated cells in metabolic acidosis and alkalosis. Am J Physiol Renal Physiol 277:F841-F849,1999 .4 N/ ~: H+ A1 |" H" J
5 x( `+ j& E# Q  I% K; h

19. Knauf, F,Yang CL,Thomson RB,Mentone SA,Giebisch G,andAronson PS. Identification of a chloride-formate exchanger expressed on the brush border membrane of renal proximal tubule cells. Proc Natl Acad Sci USA 98:9425-9430,2001 .4 Q9 A8 |% L) _

0 E% R) R5 m8 w! w# a$ v! u

20. Lohi, H,Kujala M,Kerkela E,Saarialho-Kere U,Kestila M,andKere J. Mapping of five new putative anion transporter genes in human and characterization of SLC26A6, a candidate gene for pancreatic anion exchanger. Genomics 70:102-112,2000  .* h2 Y4 L" F7 a' e8 e' t" V$ `7 }

6 z2 t+ L+ j; U! V6 \2 g

21. Madsen, KM,Verlander JW,Kim J,andTisher CC. Morphological adaptation of the collecting duct to acid-base disturbances. Kidney Int Suppl 33:S57-S63,1991 .( u4 Q7 ?( \8 J/ {; _
4 J$ ]/ z5 o- i4 U4 V  Z3 I" E; _1 B
" n, v! t7 T- s6 V. d
( X0 ]/ U  L2 M7 i) C5 L+ G
22. Melvin, JE,Park K,Richardson L,Schultheis PJ,andShull GE. Mouse down-regulated in adenoma (DRA) is an intestinal Cl /HCO 3 − exchanger and is up-regulated in colon of mice lacking the NHE3 Na /H exchanger. J Biol Chem 274:22855-22861,1999 .
% w( E# o: v. E8 X

23. Nielsen, S,DiGiovanni SR,Christensen EI,Knepper MA,andHarris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90:11663-11667,1993 .9 c! K: S/ O5 c" `' H- @% n

24. Petrovic, S,Spicer Z,Greeley T,Shull G,andSoleimani M. Novel schering and ouabain-insensitive potassium-dependent proton secretion in the mouse cortical collecting duct. Am J Physiol Renal Physiol 282:F133-F143,2002 .

% L+ ]6 f8 j( p0 I

25. Petrovic S and Schwartz GJ. In vitro acidosis reduces apicalCl/HCO3 exchanger in B-intercalated cells of rabbit cortical collectingduct (Abstract). J Am Soc Nephrol 10, A0011, 1999.: c  q6 Y7 }6 x0 i& h; s
( N+ t  T$ z) H

7 P: j& W2 s: L! k
26. Petrovic, S,Wang Z,Greeley T,Amlal H,andSoleimani M. Apical Cl /HCO 3 − exchangers pendrin and AE4 in the kidney: localization and regulation in metabolic acidosis (Abstract). J Am Soc Nephrol 12:A0036,2001.. s3 r. D; b0 G6 G8 j
2 \# Z9 [. T0 r5 e, U

; K6 i6 p, y5 Q
27. Rajendran, VM,Black J,Ardito TA,Sangan P,Alper SL,Schweinfest C,Kashgarian M,andBinder HJ. Regulation of DRA and AE1 in rat colon by dietary Na depletion. Am J Physiol Gastrointest Liver Physiol 279:G931-G942,2000 .
1 A2 `, W. a8 C4 `4 @3 K( A. R
' c" ?# x& [. K% r: k

28. Royaux, IE,Wall SM,Karniski LP,Everett LA,Suzuki K,Knepper MA,andGreen ED. 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 98:4221-4226,2001 .
6 @( D$ U4 R% B5 w8 h( m% _. F

' ^/ z' L3 T* |9 F, t
29. Sabolic, I,Brown D,Gluck SL,andAlper SL. Regulation of AE1 anion exchanger and H -ATPase in rat cortex by acute metabolic acidosis and alkalosis. Kidney Int 51:125-137,1997  .
4 u9 @; B! K# i/ E$ X: c! \

30. Satlin, LM,andSchwartz GJ. Cellular remodeling of HCO 3 − - secreting cells in rabbit renal collecting duct in response to an acidic environment. J Cell Biol 109:1279-1288,1989 .3 `% O5 ]- `) V2 B7 R( Z! F
  |. j2 |2 ]$ B: q
9 v6 ?4 Y9 o* q1 Q
* F& s9 y# t5 A$ d0 P/ o2 }
31. Schuster, VL. Function and regulation of collecting duct intercalated cells. Annu Rev Physiol 55:267-288,1993  .

32. Schwartz, GJ,Barasch J,andAl-Awqati Q. Plasticity of functional epithelial polarity. Nature 318:368-371,1985 .+ A4 O, e0 O% V1 U

! M% X1 |" U7 H! e5 }& j6 K: ^# `0 O% ^
# u: |, x* ^- a( T* s8 S" f& W3 {) E
33. Schwartz, GJ,Tsuruoka S,Vijayakumar S,Petrovic S,Mian A,andAl-Awqati Q. Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin. J Clin Invest 109:89-99,2002  .! W/ {6 [' x6 {1 o% m
& f; \" v8 Z! m' }% L* z
, d# ~9 a5 ^  t6 p+ ~

35. Soleimani, M. Molecular physiology of the renal chloride-formate exchanger. Curr Opin Nephrol Hypertens 10:677-683,2001  .

+ _4 N+ ~4 L. m. r! P, Y
/ c! F; B- S8 U2 L5 l. c' P
36. Soleimani, M,Greeley T,Petrovic S,Wang Z,Amlal H,Kopp P,andBurnham CE. Pendrin: an apical Cl /OH /HCO 3 − exchanger in the kidney cortex. Am J Physiol Renal Physiol 280:F356-F364,2001 .' j, W/ q6 X& Q

0 K1 G) i7 }9 e9 q
5 E- E" u% L; q' O3 ~" j1 a
37. Thomas, JA,Buchsbaum RN,Zimniak A,andRacker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210-2218,1979 .% E: }& _! M1 T$ K# Q+ T& D

38. Tisher, CC,Madsen KM,andVerlander JW. Structural adaptation of the collecting duct to acid-base disturbances. Contrib Nephrol 95:168-177,1991 .

39. Tsuganezawa, H,Kobayashi K,Iyori M,Araki T,Koizumi A,Watanabe S,Kaneko A,Fukao T,Monkawa T,Yoshida T,Kim DK,Kanai Y,Endou H,Hayashi M,andSaruta T. A new member of the HCO 3 − transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney. J Biol Chem 276:8180-8189,2001 ., b4 [3 Y" {- \
( A# |3 c* h7 [0 J0 n

; `5 B9 l0 [5 g1 W" J8 J+ p
40. Tsuruoka, S,andSchwartz GJ. Adaptation of rabbit cortical collecting duct HCO 3 − transport to metabolic acidosis in vitro. J Clin Invest 97:1076-1084,1996  .0 `' R& K4 ^( K& R) G" Z

2 F( P0 a& X9 l/ V6 V
41. Tsuruoka, S,Swenson ER,Petrovic S,Fujimura A,andSchwartz GJ. Role of basolateral carbonic anhydrase in proximal tubular fluid and bicarbonate absorption. Am J Physiol Renal Physiol 280:F146-F154,2001 .% j% ]/ u0 @# W# j

& }8 E0 T- z* N; M* o- t! g1 i
; e4 @2 N# O8 u; ?
42. Verlander, JW,Madsen KM,Low PS,Allen DP,andTisher CC. Immunocytochemical localization of band 3 protein in the rat collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 255:F115-F125,1988 .
8 F/ U* k" b4 a% v9 z' y

  j8 T: N: D/ j$ G4 s
43. Verlander, JW,Madsen KM,andTisher CC. Structural and functional features of proton and bicarbonate transport in the rat collecting duct. Semin Nephrol 11:465-477,1991  .: \: T4 x9 g8 Q

44. Wang, Z,Petrovic S,Mann E,andSoleimani M. Identification of an apical Cl /HCO 3 − exchanger in the small intestine. Am J Physiol Gastrointest Liver Physiol 282:G573-G579,2002 .6 I# m" X7 v2 @+ q7 [  E

1 Z2 h+ {2 b# h' o0 ^+ Z

45. Weiner, ID,andHamm LL. Use of fluorescent dye BCECF to measure intracellular pH in cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 257:F957-F964,1989.
  t/ C6 G5 Y$ s, D* X

46. Weiner, ID,andHamm LL. Regulation of Cl /HCO 3 − exchange in the rabbit cortical collecting tubule. J Clin Invest 87:1553-1558,1991  .
/ C! h3 `& Q: m! G& ?5 D+ \/ |
; y8 Y6 `* D5 r1 s& h5 ?
+ f, Y, P8 a% P* \) Z
47. Weiner, ID,Weill AE,andNew AR. Distribution of Cl /HCO 3 − exchange and intercalated cells in rabbit cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 267:F952-F964,1994 .

收藏0 分享0 顶0 踩0