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作者：Young-Hee Kim, Jin Kim, A. S. Verkman, and Kirsten M. Madsen作者单位：1 Department of Medicine, University of Florida,Gainesville, Florida 32610-0215; 2 Department ofAnatomy, Catholic University Medical College, Seoul 137-70 Korea; and 3 Departments of Medicine and Physiology,Cardiovascular Research Institute, University of California, San Francisco,California 94143-0
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" r# O3 P: i5 x# j9 \4 v 【摘要】$ T- |( E, I& k0 N% g
Phenotype analysis has demonstrated that aquaporin-1 (AQP1) null mice arepolyuric and manifest a urinary concentrating defect because of an inabilityto create a hypertonic medullary interstitium. We report here that deletion ofAQP1 is also associated with a decrease in urinary pH from 6.15 ± (SE)0.1 to 5.63 ± 0.07. To explore the mechanism of the decrease in urinarypH, we examined the expression of H -ATPase in kidneys of AQP1 nullmice. There was strong labeling for H -ATPase in intercalated cellsand proximal tubule cells in both AQP1 null and wild-type mice. StrongH -ATPase immunostaining was also present in the apical plasmamembrane of inner medullary collecting duct (IMCD) cells in AQP1 null mice,whereas no H -ATPase labeling was observed in IMCD cells inwild-type mice. In addition, there was an increase in the prevalence of type Aintercalated cells in the IMCD of AQP1 null mice, suggesting that the deletionof intercalated cells from the IMCD, which normally occurs during postnatalkidney development, was impaired. Western blot analysis ofH -ATPase expression in the different regions of the kidneydemonstrated a significant increase in H -ATPase protein in theinner medulla of AQP1 null mice compared with wild-type mice. There were nochanges in H -ATPase expression in the cortex or outer medulla.These results represent the first demonstration of apical H -ATPaseimmunoreactivity in IMCD cells in vivo and suggest that the decrease inurinary pH observed in AQP1 null mice is due to upregulation ofH -ATPase in the IMCD. The induction of H -ATPaseexpression in IMCD cells of AQP1 null mice may be related to the chronicallylow interstitial osmolality in these animals. The challenge will be toidentify the molecular signal(s) responsible for the de novoH -ATPase expression.
' n( z. z% a% [ 【关键词】 urine acidification intercalated cells immunohistochemistry
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' r8 P1 _. Q' SAQUAPORIN -1 ( AQP 1), A WATER -transporting protein, is expressed in the apical and basolateral plasma membranes of the proximal tubule, thedescending thin limb of Henle's loop, and the microvascular endothelium of thedescending vasa recta ( 12, 19, 20 ). Recent studies in AQP1null mice generated by targeted gene disruption have established the role ofAQP1 as a major water channel in the kidney( 16, 25, 32 ). Phenotype analysis hasdemonstrated that AQP1 null mice are polyuric and manifest a urinaryconcentrating defect because of an inability to create a hypertonic medullary interstitium ( 9, 16, 23, 32 ) as well as defectiveisosmolar fluid absorption by the proximal tubule( 25, 31 ). Further analysis of urinecomposition, reported in this study, revealed a significantly decreasedurinary pH in AQP1 null mice; however, the mechanism behind the increasedacidity of the urine is unclear.
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2 D9 H0 r8 O. f6 g; _. aThe collecting duct plays an important role in acid-base transport in thekidney and is the main site of urine acidification ( 3 ). Acid secretion occursalong the entire collecting duct, whereas bicarbonate secretion has beendemonstrated only in the cortical collecting duct (CCD). There is convincingevidence that intercalated cells are responsible for acid-base transport inthe collecting duct. At least two types of intercalated cells, type A and typeB, are present in the collecting duct ( 2, 13, 26, 29, 35 ). Type A intercalated cellssecrete protons and absorb bicarbonate. Proton secretion is mediated, at leastin part, by a vacuolar-type H -ATPase that is located in the apicalplasma membrane and apical tubulovesicles( 7, 8, 13, 34 ). Bicarbonate absorptionoccurs via AE1, a truncated form of the erythrocyteCl - /HCO 3 - exchanger( 13, 26, 29, 33 ). Type B intercalated cellsare responsible for bicarbonate secretion in the CCD( 39 ). There is also evidencethat acid secretion in the collecting duct can occur via anH -K -ATPase that has been demonstrated in both type Aand type B intercalated cells( 36, 40, 41 ).- O2 X( U; `! ^: l e2 B0 S
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Intercalated cells gradually disappear in the inner medullary collectingduct (IMCD) and are not observed in the terminal one-half to two-thirds of theIMCD. However, there is evidence that acid secretion occurs along the entireIMCD, indicating that IMCD cells may also be capable of acid secretion( 1 ). Studies usingmicrocatheterization and micropuncture techniques have demonstrated a decreasein urinary pH along the IMCD ( 10, 24, 30 ). Both acute and chronicmetabolic acidosis is associated with increased urine acidification along theIMCD ( 6, 10 ), whereas acidification isabolished during acute and chronic alkalosis( 5 ). Subsequent studiesdemonstrated net acid secretion in isolated IMCD segments from normal rats and reported that acid secretion was significantly increased in IMCD segments fromammonium chloride-loaded rats( 37 ). The cellular mechanismof acid secretion in the IMCD is not known with certainty. However, there isevidence from studies in cultured IMCD cells that acid secretion may bemediated by both the bafilomycin-sensitive vacuolar-type H -ATPase( 27 ) and theSCH-28080-sensitive H -K -ATPase( 22 ). Studies in the isolatedperfused IMCD from rats have also reported that acid secretion is mediated byan H -K -ATPase in the terminal IMCD( 38 ). Although in vitrostudies have provided evidence that acid secretion in IMCD cells can bemediated by H -ATPase as well asH -K -ATPase, immunohistochemical studies have failed todemonstrate expression of either protein in IMCD cells in vivo under anyexperimental conditions.5 F0 {* f' u; w) ^9 f d/ x
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To explore the mechanism of the decrease in urinary pH observed in micedeficient in AQP1, we examined the expression of the vacuolarH -ATPase in kidneys of AQP1 null and wild-type mice by light andelectron microscopic immunocytochemistry and Western blot analysis. Ourresults demonstrate a significant increase in the expression ofH -ATPase in the inner medulla with induction ofH -ATPase in the apical plasma membrane of IMCD cells and increasedprevalence of type A intercalated cells in the IMCD of AQP1-deficient micecompared with wild-type mice. Based on these observations, we propose that theinability to create a hypertonic medullary interstitium may play a role in the expression of H -ATPase in IMCD cells and interfere with the deletion of intercalated cells from the IMCD that normally occurs duringpostnatal kidney development.1 t. p) R" V8 @ l
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Animals and Tissue Preservation
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! |- `9 K6 S: p- g, f) S/ c) hThe transgenic AQP1-deficient mice that were generated by targeted genedisruption have been characterized in detail in previous studies( 16 ). The pH of freshlyspontaneously voided urine specimens from six AQP1 null and six wild-typemice, 4-6 wk of age, was measured with pH electrodes. Animal experiments wereperformed in accordance with the National Institutes of Health Guide forthe Care and Use of Laboratory Animals (Washington, DC: National AcademyPress, 1996) and were approved by the University of California, San Francisco,and University of Florida Institutional Animal Care and Use Committees.
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' {0 p2 z. p: a5 C! FA total of eight mice, four AQP1 ( / ) and four AQP1 (-/-), were used forstudies of H -ATPase expression by immunohistochemistry and Westernblot analysis. Additional animals were used for double-immunolabelingexperiments. The animals were anesthetized with an intraperitoneal injectionof pentobarbital sodium (50 mg/kg body wt). The kidneys were perfused brieflywith PBS to rinse away all blood. From animals used for studies ofH -ATPase expression, the right kidney was excised and processedfor immunoblot analysis. This was followed by perfusion of the left kidneywith a periodate-lysine-2% paraformaldehyde (PLP) solution for 10 min. Inanimals used only for immunohistochemical studies, both kidneys were preservedby perfusion with PLP. The kidneys were removed and cut into 1- to 2-mm-thickslices that were fixed additionally by immersion in the same fixative for 2 h at room temperature and then overnight at 4°C. Sections of tissue were cuttransversely through the entire kidney on a vibratome (Pelco 101, Sectioningseries 1000, Ted Pella, Redding, CA) at a thickness of 50 µm and processedfor immunohistochemical studies using a horseradish peroxidase preembedding technique.
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5 U7 P/ k$ k0 N0 QAntibodies
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Rabbit polyclonal antibodies were used in all experiments. H -ATPase immunoreactivity was detected by immunohistochemical andimmunoblot analysis using an antibody against the 70-kDa subunit of thevacuolar H -ATPase (courtesy of Dr. Dennis Stone, Univ. of TexasSouthwestern, Dallas, TX). IMCD cells were identified with an antibody to AQP4(courtesy of Dr. Mark A. Knepper, National Institutes of Health, Bethesda,MD). Type A intercalated cells were identified with an antibody against AE1(courtesy of Dr. Philip S. Low, Purdue Univ., West Lafayette, IN). Lack ofAQP1 immunoreactivity in AQP1 null mice was confirmed using an antibodyagainst AQP1 (provided by Dr. M. A. Knepper).9 j1 B) s, ^! }" ]; h" l
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Immunohistochemistry
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Preembedding method. Sections of PLP-fixed tissue were cut transversely through the kidney on a vibratome at a thickness of 50 µm andprocessed for immunohistochemistry using an indirect immunoperoxidase method.All sections were washed three times with 50 mM NH 4 Cl in PBS for 15min. Before incubation with the primary antibodies, the sections wereincubated for 3 h with PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin ( solution A ). The tissue sections were then incubated overnight at4°C in a solution of polyclonal antibodies against H -ATPase (1:2,000), AQP1 (1:500), AQP4 (1:300), or AE1 (1:2,000) in PBS containing 1%BSA ( solution B ). After several washes with PBS containing 0.1% BSA,0.05% saponin, and 0.2% gelatin ( solution C ), the tissue sectionswere incubated for 2 h in peroxidase-conjugated donkey anti-rabbit IgG, Fabfragment (JacksonImmunoResearch Labs) diluted 1:100 in solution B.The sections were then rinsed, first in solution C and subsequentlyin 0.05 M Tris buffer, pH 7.6. For the detection of horseradish peroxidase,the sections were incubated in 0.1% 3,3'-diaminobenzidine (brown stain) in 0.05 M Tris buffer for 5 min, after which H 2 O 2 wasadded to a final concentration of 0.01% and the incubation was continued for10 min. After being washed three times with 0.05 M Tris buffer, the sectionswere dehydrated in a graded series of ethanol and embedded in TAAB resin. Fromall animals, 50-µm-thick vibratome sections cut through the entire kidneywere mounted in TAAB between polyethylene vinyl sheets. For the observation athigh magnification, sections from the inner medulla were excised and gluedonto empty blocks of TAAB, and 1.5-µm semithin sections were cut for lightmicroscopy. The sections were photographed on an Olympus Photomicroscopeequipped with differential-interference contrast optics.
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Double immunolabeling for AQP4 and H -ATPase. To identify the cells labeled with H -ATPase in the IMCD, doublelabeling was performed with antibodies against H -ATPase and AQP4,a marker of principal cells and IMCD cells. From the flat-embedded50-µm-thick sections processed for immunolabeling of AQP4, sections from the inner medulla were excised and glued onto empty blocks of TAAB 812, andconsecutive 1.5-µm sections were cut for postembedding immunolabeling. Thesections were treated for 15 min with a saturated solution of sodium hydroxidein absolute ethanol to remove the resin. After three brief rinses in absolute ethanol, the sections were hydrated with graded ethanol and rinsed in tapwater. The sections were rinsed with PBS, incubated in normal donkey serum for30 min, and subsequently incubated overnight with antibody againstH -ATPase (1:1,000) at 4°C. The sections were rinsed with PBS,incubated for 2 h in peroxidase-conjugated donkey anti-rabbit IgG, Fabfragment, and washed again with PBS. For detection of H -ATPase,Vector SG (Vector Laboratories) was used as the chromogen to produce a bluegray label, which is easily distinguishable from the brown label produced by 3,3'-diaminobenzidine used in the preembedding procedure for detectionof AQP4. The sections were washed with distilled water, dehydrated with gradedethanol and xylene, mounted in Canada balsam, and examined by lightmicroscopy.
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Transmission Electron Microscopy3 H4 t2 ?2 L4 R% g F' l( w
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Tissue slices immunostained for H -ATPase were fixed in 2% glutaraldehyde in 0.1 M Tyrode buffer for 1 h at 4°C and washed twice for10 min, first with 0.1 M Tyrode buffer and then with 0.1 M sodium cacodylatebuffer. This was followed by postfixation in 1% osmium tetroxide in 0.1 Msodium cacodylate buffer for 1 h at 4°C. After being rinsed in 0.1 Msodium cacodylate buffer, the tissue was dehydrated in a graded series ofalcohol and propylene oxide and embedded in TAAB resin. Ultrathin sectionswere stained with lead citrate and photographed with a Zeiss 10 A transmissionelectron microscope.
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Immunoblot Analysis
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Kidneys from four AQP1 ( / ) and four AQP1 (-/-) mice were excised andseparated into cortex, outer medulla, and inner medulla. The tissue washomogenized in a solution containing 10 mM Tris·HCl (pH 7.6), 150 mMNaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.5 mM EDTA, 0.1% SDS, andfreshly added leupeptin (5 mg/ml) and 100 mM phenylmethylsulfonyl fluoride.Homogenates were centrifuged at 12,000 g for 15 min at 4°C, andprotein concentrations were determined on supernatants using the BCA proteinassay re-agent (Pierce, Rockford, IL). The proteins were resolved by SDS-PAGE,electrophoretically transferred onto nitrocellulose membranes, and then probedwith antibodies to H -ATPase. The immune complexes were detectedwith horseradish peroxidase-conjugated goat anti-rabbit IgG, and the sites of antibody-antigen reaction were visualized using a chemiluminescence detectionkit (Amersham Life Science, Buckinghamshire, UK).5 f: C; t- @/ `
' U6 i* |$ C# r4 IStatistical Analysis0 a% ^& j) N& h5 k: {$ X- U) a
3 L% a5 o# D' P4 g6 L; G3 CTo quantify the level of expression of H -ATPase,autoradiographs were scanned on a Hewlett-Packard Scanjet 4C using Deskscan IIsoftware, and densitometry was performed using National Institutes of HealthIMAGE 1.60 software. The results are presented as means ± SE. Thestatistical significance of the difference in H -ATPase expressionbetween AQP1 (-/-) and AQP1 ( / ) mice was assessed using Student's unpaired t -test. P values 5 w4 F3 F) Q6 E6 L
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RESULTS
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Determination of pH in the spontaneously voided urine samples demonstrateda significantly lower pH in urine from AQP1-deficient mice than in samplesfrom wild-type mice (5.63 ± 0.07 vs. 6.15 ± 0.1; P
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Fig. 1. Diagram showing the pH of freshly voided urine samples from aquaporin-1(AQP1) null (AQP1 -/-; ) and wild-type (AQP1 / ; ) mice. There wassignificantly decreased urinary pH in association with AQP1 deletion. Valuesare means ± SE; n = 6/group. * P
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Immunohistochemistry) u- x& T* j" E- H* C6 M. w
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As illustrated in Fig. 2,there was no AQP1 immunoreactivity in kidneys of AQP1 knockout mice, whereasAQP1 was expressed in the proximal tubule, thin descending limb, and vasarecta of wild-type mice, as reported in previous studies( 12, 19, 20 ).
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$ H6 q- Z/ C9 iFig. 2. Light micrographs of 50-µm-thick sections from AQP1 ( / ) and AQP1 (-/-)mouse kidney ( A and B, respectively) illustratingimmunostaining for AQP1. AQP1 was strongly expressed in the proximal tubule,descending thin limb of Henle's loop, and descending vasa recta in kidney fromAQP1 ( / ) mouse, but there was no labeling in the kidney of AQP1 (-/-) mouse.Co, cortex; OS, outer stripe of outer medulla; IS, inner stripe of outermedulla. Magnification: x 12 ( A and B ).
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. C3 v% q' a6 o0 y& K PImmunostaining for H -ATPase was observed in proximal tubule cells and in intercalated cells in the connecting tubule and collecting ductin both wild-type and AQP1 knockout mice( Fig. 3 ). As the collectingduct descended toward the renal papilla, H -ATPase-positive cellsdecreased in number in wild-type mice, and there was no H -ATPaseimmunolabeling from the middle part of the IMCD( Fig. 3 A ). However, inAQP1 knockout mice, strong H -ATPase immunoreactivity was observedin IMCD cells, and the intensity of labeling increased in the terminal papilla ( Fig. 3 B ).Furthermore, intercalated cells were also observed in the middle part of theIMCD ( Fig. 3 B ), andthe number of H -ATPase-positive intercalated cells appeared to beincreased in the initial part of the IMCD in AQP1 null mice compared with wild-type mice. Higher magnification light microscopy revealed thatH -ATPase immunostaining of IMCD cells in AQP1 null mice waslocated in the apical plasma membrane, and there was no H -ATPaseimmunolabeling of the basolateral plasma membrane ( Fig. 4 ). There was a gradualincrease in the intensity of H -ATPase immunoreactivity from themiddle part of the IMCD to the tip of the renal papilla( Fig. 4, B and C ). However, there was no labeling of principal cells inthe initial IMCD ( Fig.4 A ).
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Fig. 3. Light micrographs of 50-µm-thick sections from AQP1 ( / ) and AQP1 (-/-)mouse kidney ( A and B, respectively) illustratingimmunostaining for H -ATPase. H -ATPase was stronglyexpressed in the collecting duct and connecting tubule, but there was lesslabeling in the proximal tubule in both AQP1 ( / ) and AQP1 (-/-) mousekidney. In the collecting duct of AQP1 ( / ) mouse kidney,H -ATPase was mainly expressed in cortical and outer medullarysegments, and there was no immunolabeling in the middle and terminal part ofthe inner medullary collecting duct (IMCD; A ). However, in the AQP1(-/-) mouse, there was strong H -ATPase immunoreactivity in allsegments of the collecting duct, including the IMCD ( B ), and theintensity of AQP1 immunolabeling gradually increased from the middle of theIMCD toward the tip of the papilla. In addition, there was an increase in theprevalence of H -ATPase-positive type A intercalated cells in themiddle part of the IMCD from the AQP1 (-/-) mouse. However, there were nodifferences in the intensity of H -ATPase immunostaining ofintercalated cells between AQP1 (-/-) and AQP1 ( / ) mice. OM, outer medulla;IM, inner medulla. Magnification: x 12 ( A and B ).( j8 M0 S) y! V4 M
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Fig. 4. Light micrographs of 50-µm-thick sections of initial ( A;IMCDi), middle ( B; IMCDm), and terminal part ( C; IMCDt) ofrenal IMCD from AQP1 (-/-) mouse kidney. There was strong H -ATPaseimmunoreactivity in type A intercalated cells (arrows) but no or weakimmunolabeling of principal cells in IMCDi ( A ). From IMCDm( B ), H -ATPase immunoreactivity was observed in the apicalmembrane of IMCD cells and gradually increased toward IMCDt ( C ).Magnification: x 400., S& ^' L8 s, }, k& i6 ?& G1 C/ h
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The expression of H -ATPase in the apical plasma membrane was confirmed by transmission electron microscopy, which also revealed strikingchanges in the configuration of IMCD cells in AQP1 null mice( Fig. 5 ). The junctionalcomplex appeared to be transposed toward the basement membrane, leading to adecrease in the height of the intercellular space and the lateral plasma membrane and a corresponding increase in the apical membrane. H -ATPase immunostaining was observed on the entire apical plasma membrane of the IMCD cells, but there was no labeling of the basolateralmembrane.
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Fig. 5. Transmission electron microscopic localization of H -ATPase inthe IMCDt of AQP1 (-/-) mouse kidney. A : H -ATPaseimmunolabeling (arrowheads in B ) was observed in the apical plasmamembrane of IMCD cells. Note an H -ATPase-negative cell thatexhibits the characteristics of principal cells (asterisk). B : highermagnification of the area indicated by rectangle in A. Note that inthe AQP1 (-/-) mouse, the location of the tight junction (arrows) was lowerthan that in the control mouse, resulting in an extension of the apical plasmamembrane and a reduction in the length of the lateral plasma membrane.Magnification: x 3,600 ( A ); x 11,000 ( B ).- u1 U9 }+ L4 [5 w$ e! `! E
8 a( R; [& A' T( A8 gTo confirm that the labeled cells were IMCD cells, we performed doublelabeling with antibodies against AQP4, which is expressed in the basolateralplasma membrane of IMCD cells, and H -ATPase. The resultsdemonstrated that in the AQP1 null mice, H -ATPase immunoreactivitywas located in the apical plasma membrane of AQP4-positive cells in theterminal half of the IMCD, thus identifying them as IMCD cells( Fig. 6 A, and B ). There was no H -ATPase immunoreactivity inAQP4-positive cells in wild-type mice (not shown). Intercalated cells withstrong apical H -ATPase immunostaining, but no AQP4 immunolabeling,were also present in the middle portion of the IMCD in AQP1 null mice( Fig. 6 A ). Labelingwith antibodies against AE1, the basolateral anion exchanger, identified theintercalated cells in the IMCD as type A intercalated cells( Fig. 7 ).
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$ Z% q( R& u0 p8 CFig. 6. Light micrographs of 1.5-µm-thick sections of IMCDm ( A ) andIMCDt ( B ) from AQP1 (-/-) mouse kidney illustrating doubleimmunolabeling for AQP4 (brown) and H -ATPase (blue gray). Therewas H -ATPase immunoreactivity in the apical membrane ofAQP4-positive IMCD cells (arrows) from the middle ( A ) to the terminalpart of the renal papilla ( B ). Note intercalated cells (asterisk)with H -ATPase but without AQP4 immunolabeling. Bar = 10 µm.Magnification: x 400.1 ?5 O9 l5 K1 y* L) t
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Fig. 7. Light micrographs of 50-µm-thick sections from AQP1 ( / ) and AQP1 (-/-)mouse kidney ( A and B and C, respectively)illustrating immunostaining for Cl - /HCO 3 - exchanger (AE1). A : AE1 expression was limited to intercalated cellsin IMCDi, and there was no AE1 immunolabeling in the remaining part of thepapilla in AQP1 ( / ) mouse kidney. However, in AQP1 (-/-) mouse, cells withstrong AE1 immunostaining were present even in IMCDm ( B ). Highmagnification revealed AE1 labeling of the basolateral plasma membrane of theintercalated cells (arrows) in this region ( C ). Magnification: x 30 ( A ); x 55 ( B ); x 450 ( C ).
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! Y3 a1 l* c6 b0 ?Immunoblot Analysis$ S |0 F( `. w, Q4 N
6 j: t. e7 }8 o' C7 d( Z4 s( j. ^To quantify the changes in H -ATPase expression in AQP1 null mice, we carried out immunoblot analysis with kidney homogenates in which thecortex, outer medulla, and inner medulla were separated and probed withantibodies to H -ATPase. There was a statistically significantincrease in H -ATPase expression in the inner medulla of AQP1knockout mice (299 ± 19) compared with wild-type animals (153 ±94; P
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; i5 q9 g7 T F! I% K! RFig. 8. Western blot analysis demonstrating H -ATPase expression in Co,OM, and IM of kidneys from AQP1 ( / ) and AQP1 (-/-) mouse kidneys. A : SDS-PAGE was performed using 10% polyacrylamide gels, and 10 µgof protein were loaded in each lane. There was a prominent band at 70 kDa inboth AQP1 ( / ) and AQP1 (-/-) mouse kidneys. Note the marked increase inH -ATPase protein in IM of AQP1 (-/-) mice compared with AQP1 ( / )mice. No changes were observed in Co and OM. B : immunoblottingdensitometry of H -ATPase levels in Co, OM, and IM. There was asignificant difference in the expression of H -ATPase protein inthe IM between AQP1 ( / ) and AQP1 (-/-) mice ( * P # X+ f' f% ]9 \
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DISCUSSION* d0 ^6 ~0 U# z$ H# h# Z
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In this study, we demonstrated that deletion of AQP1 is associated with asignificant decrease in urinary pH. Because acid secretion in the collectingduct is the main determinant of urinary pH, and is to a large degree mediatedby a vacuolar-type H -ATPase, we determined the expression ofH -ATPase in the kidney of AQP1 (-/-) and AQP1 ( / ) mice.
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% L2 j! z6 _" d2 _& [2 {! GOur results demonstrate a significant increase in the expression ofH -ATPase in the inner medulla of AQP1 (-/-) mice compared withAQP1 ( / ) animals. The increase in H -ATPase expression was due toan induction of H -ATPase in the apical plasma membrane of IMCDcells and an increase in the prevalence of type A intercalated cells in theIMCD. There were no changes in H -ATPase expression in eithercortex or outer medulla of AQP1 null mice. These results represent the firstdemonstration of apical H -ATPase immunoreactivity in IMCD cells invivo and suggest that the decrease in urinary pH observed in AQP1 null mice isdue to upregulation of H -ATPase in the IMCD.
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9 c- ?/ ^, N2 W0 I$ d( p+ Q" \4 ?Although numerous immunohistochemical studies have examined the expressionand distribution of H -ATPase in the collecting duct,H -ATPase immunoreactivity has not been observed in IMCD cells invivo in either normal or acidotic conditions. However, it is well establishedfrom microcatheterization and micropuncture studies of the papillarycollecting duct that acid secretion occurs along the entire IMCD( 10, 24, 30 ) and is increased in bothacute and chronic metabolic acidosis( 6, 10 ). In vitro studies incultured IMCD cells have demonstrated the presence of bafilomycin- orSCH-28080-sensitive acid secretion in these cells, indicating thatH -ATPase as well as H -K -ATPase can beexpressed in IMCD cells in vitro( 22, 27 ). Interestingly, moststudies have reported only one of the two transporters in cultured IMCD cells,suggesting that the expression of both H -ATPase andH -K -ATPase may be dependent on the in vitro cultureconditions or on the site of origin of the IMCD cells in the renal papilla.Thus discrepancies existed between the results of immunohistochemical andfunctional studies. However, the present demonstration of H -ATPaseimmunoreactivity in IMCD cells indicates that under certain conditions, acidsecretion in the terminal IMCD may be mediated by a vacuolar-typeH -ATPase located in the apical plasma membrane of IMCD cells.Previous studies by Stuart-Tilley et al.( 28 ) have demonstratedexpression of the anion exchanger AE2 in the basolateral plasma membrane ofIMCD cells in normal mouse kidney, suggesting that bicarbonate absorption inthese cells is mediated by AE2.4 N) K" ]: U! }( G
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The mechanism of the increased expression of H -ATPase in the inner medulla and the induction of H -ATPase in the apical plasma membrane of the IMCD cells is not known. However, it is well established thatAQP1-deficient mice are unable to generate a hypertonic medullary interstitium( 16, 32 ). The low interstitial osmolality is believed to be due to a disruption of the countercurrent multiplication mechanism because of decreased water permeability of thedescending limb of Henle's loop and the descending vasa recta( 9, 23 ). Thus it is tempting tospeculate that the inappropriately low tonicity in the renal medulla may play a role in the induction of H -ATPase in the IMCD cells.8 }0 c& a/ R8 Q6 n
, b5 {& k& U" Z$ d' c7 o+ YA previous study by Amlal and co-workers( 4 ) has provided evidence thatH -ATPase activity is regulated by hypotonicity. In vitro studiesin cultured IMCD cells demonstrated that the rate of sodium-independent pHrecovery in response to an acid load was significantly greater in a hypotonicsolution than in an isotonic solution. The pH recovery was independent of thepresence of potassium and was inhibited by N -ethylmaleimide,indicating that it was mediated by vacuolar H -ATPase. When IMCDcells were incubated and assayed in a hypertonic solution, the rate of pHrecovery was decreased, indicating that H -ATPase activity isdecreased by hypertonicity ( 4 ).Whether a change in interstitial tonicity has a direct effect onH -ATPase protein expression remains to be established. If ahypertonic environment inhibited not only H -ATPase activity butalso protein expression, it might prevent the expression ofH -ATPase in IMCD cells under normal in vivo conditions and thusexplain the discrepancy between the results of in vivo immunohistochemicalstudies and studies of IMCD cells in vitro.$ E- w1 ^* X6 n7 N/ I6 _4 x C
2 s: s4 d: Y' t+ `3 y0 y vInterestingly, although the deletion of AQP1 was associated with inductionof H -ATPase and the appearance of strong immunoreactivity in theapical plasma membrane of IMCD cells, there were no changes in the level ofH -ATPase expression in intercalated cells in any segments of thecollecting duct. These results indicate that the mechanism of regulation ofH -ATPase expression is different in IMCD cells and intercalatedcells. In this regard, it is noteworthy that V 2 vasopressinreceptors are present in IMCD cells( 21 ), but not in intercalatedcells, thus raising the possibility that changes in vasopressin levels mightplay a role in the regulation of H -ATPase expression in IMCDcells., a5 i& g+ m, I0 Z
+ D2 L) ?; z8 n4 ]1 XAlthough there were no changes in the intensity of H -ATPase immunolabeling in intercalated cells, the prevalence of type A intercalatedcells in the IMCD was increased. In wild-type animals, type A intercalatedcells were present only in the initial part of the IMCD. However, inAQP1-deficient mice, type A intercalated cells were observed also in themiddle part of the IMCD. It is well known that in the neonatal kidney, intercalated cells are found along the entire collecting duct. After birth,during the time of development of the renal papilla and a hypertonic medullaryinterstitium, intercalated cells are deleted from the terminal one-half totwo-thirds of the IMCD and only IMCD cells remain in this part of thecollecting duct ( 11, 14 ). The demonstration in thisstudy that intercalated cells remain in the middle segment of the IMCDsuggests that local changes in the renal papilla associated with AQP1deficiency interfere with the normal deletion of intercalated cells from theIMCD. There is evidence from recent studies in cultured IMCD cells thatosmotic stress causes DNA damage and cell death ( 15, 18 ). Although IMCD cellsappear to be quite tolerant of hyperosmotic conditions in vivo, it is quitepossible that intercalated cells, which are rich in mitochondria, are more sensitive to osmotic stress. It is noteworthy that hypertonicity has beenshown to cause mitochondrial dysfunction, which can initiate apoptotic celldeath ( 17 ).! O$ e9 B- n S* k
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In summary, the present study demonstrates that AQP1 gene deletion isassociated with a significant increase in the expression ofH -ATPase in the inner medulla, increased prevalence of intercalated cells in the IMCD, and induction of H -ATPase in theapical plasma membrane of IMCD cells in the mouse kidney. The challenge willbe to identify the molecular signal(s) responsible for the induction andregulation of H -ATPase expression in the IMCD cells.
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. @5 ]/ h% r, T+ Z1 D. o2 P2 m K+ W* LACKNOWLEDGMENTS7 G: r+ F* i$ u. ?) l$ P, X) U- D
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The authors thank Dr. Søren Nielsen, Aarhus University, Aarhus,Denmark, and Dr. C. Craig Tisher, University of Florida, Gainesville, FL, foradvice and support during this project.
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% x/ [6 t% p" m" rPresent address of Y.-H. Kim: The Water and Salt Research Center, Univ. ofAarhus, DK-8000 Aarhus C, Denmark.# {+ c) T, I' J# N1 c3 ^% m( E
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