|
- 积分
- 0
- 威望
- 0
- 包包
- 0
|
作者：Jill W.Verlander, R. TylerMiller, Amy E.Frank, InesE.Royaux, Young-HeeKim, I. DavidWeiner,作者单位：1 University of Florida College of Medicine, and North Florida/South Georgia Veterans Health System,Gainesville, Florida 32610; Case Western ReserveUniversity and Cleveland Veterans Affairs Medical Center, Cleveland,Ohio 44106; and Genome Technology Branch,National Human Genome Research Institute, N
' v2 j" U+ D# h+ S, p, N0 {
! ^* b) X$ m/ u 6 C$ h: c' w/ t( K9 C. v" U
2 v2 v i* ^2 x) L( t 3 H4 X9 y& L' {0 |$ }( z4 v
; W- X5 a2 ~ j6 U% j- _/ A
* B7 \6 F$ C( v) R
2 p; K0 _* r% j ! }- r! J6 }2 { A5 b: V# F% }6 {
0 l+ Z- `9 k O! f" C0 [- s
; {% B3 H0 Q, Y/ v
3 `2 Z5 G/ L3 f- a' |; m. f 6 O0 `3 l2 G0 y# Q0 x( e7 y
【摘要】4 j5 q* d0 s; t* {% G: X
Ammoniais both produced and transported by renal epithelial cells, and itregulates renal ion transport. Recent studies have identified a familyof putative ammonium transporters; mRNA for two members of this family,Rh B-glycoprotein (RhBG) and Rh C-glycoprotein (RhCG), is expressed inthe kidney. The purpose of this study was to determine the cellularlocation of RhBG and RhCG protein in the mouse kidney. We generatedRhBG- and RhCG-specific anti-peptide antibodies. Immunoblot analysisconfirmed that both proteins were expressed in the mouse kidney. RhBGlocalization with immunohistochemistry revealed discrete basolaterallabeling in the connecting segment (CNT) and in the majority of initialcollecting tubule (ICT) and cortical collecting duct (CCD) cells. Inthe outer medullary collecting duct (OMCD) and inner medullarycollecting duct (IMCD) only a subpopulation of cells exhibitedbasolateral immunoreactivity. Colocalization of RhBG with carbonicanhydrase II, the thiazide-sensitive transporter, and the anionexchangers AE1 and pendrin demonstrated RhBG immunoreactivity in allCNT cells and all CCD and ICT principal cells. In the ICT and CCD,basolateral RhBG immunoreactivity is also present in A-typeintercalated cells but not in pendrin-positive CCD intercalated cells.In the OMCD and IMCD, only intercalated cells exhibit RhBGimmunoreactivity. Immunoreactivity for a second putative ammoniumtransporter, RhCG, was present in the apical region of cells withalmost the same distribution as RhBG. However, RhCG immunoreactivitywas present in all CCD cells, and it was present in outer stripe OMCDprincipal cells, in addition to OMCD and IMCD intercalated cells. Thusthe majority of RhBG and RhCG protein expression is present in the sameepithelial cell types in the CNT and collecting duct but with oppositepolarity. These findings suggest that RhBG and RhCG may play importantand cell-specific roles in ammonium transport and signaling in theseregions of the kidney.
" l4 ]% E0 q( J: N' y: F 【关键词】 Rh Bglycoprotein Rh Cglycoprotein collecting duct intercalatedcell principal cell immunohistochemistry connecting segment% p. p% a% B4 I
INTRODUCTION \. w" i4 y. q6 u
0 Z& H. g7 ?0 z2 ~
AMMONIUM TRANSPORT AND SIGNALING are importantfor the renal regulation of electrolytehomeostasis. 1 Briefly,ammonium is produced in the proximal tubule, preferentially secretedinto the luminal fluid, reabsorbed via specific membrane proteins inthe medullary thick ascending limb of the loop of Henle, and thensecreted into the luminal fluid by the collecting duct ( 4, 18 ). Production and excretion of ammonium result in productionof equimolar amounts of new bicarbonate molecules, which is importantfor replenishing base consumed by endogenous and exogenous acidproduction ( 4, 18 ). In addition to being a metabolicproduct and transported molecule, ammonia also regulates collectingduct sodium, potassium, and acid-base transport ( 6, 7, 9, 11, 40 ). Accordingly, understanding ammonium transport mechanismsand the mechanisms through which ammonia might alter collecting duction transport are important.; ]3 t7 {8 }, U
9 B/ f a1 R( y0 E& {, I1 D
A novel ammonium (NH 4 + ) transporter family of proteinswas recently identified in yeast and plants ( 29, 32 ) andis present in species throughout nature, including mammals ( 13, 14, 27 ). These proteins are glycosylated, integral membraneproteins that possess 12 predicted membrane-spanning segments. Whenexpressed in heterologous expression systems, they transport ammoniumand its analog, methylammonium ( 13, 27 ). They can alsofunction, at least in yeast, as ammonia sensors, altering cellularfunction in response to changes in extracellular ammonia ( 24, 25 )." W5 C, N8 @3 s ?. ~
5 u: ~9 U$ v. G
Three mammalian members of this family, Rh A-glycoprotein (RhAG)( 26 ), Rh B-glycoprotein (RhBG) ( 23 ), and RhC-glycoprotein (RhCG) ( 22, 26 ), have been identified. Theyexhibit substantial homology to each other and to the erythrocyte Rhfactor, a protein well recognized as an antigen in transfusion medicinebut whose function in erythrocyte membranes is not known ( 12, 14 ). RhAG protein is expressed in the erythrocyte membrane,where it may contribute to erythrocyte ammonium transport( 21 ). RhBG mRNA is expressed in liver, kidney, and skin( 23 ), and RhCG mRNA is expressed in kidney, testes, andbrain ( 22 ). Thus both RhBG and RhCG mRNA are expressed inorgans where ammonium transport is a critical function.- a" L9 C2 V6 Z5 U
& |, L8 I6 \. K p6 ]9 ?# fThe purpose of the present studies was to determine whether RhBG andRhCG proteins are expressed in the mammalian kidney and, if so, toidentify their specific cellular locations by using light microscopicimmunolocalization techniques. Anti-peptide antibodies to cytoplasmicregions of the COOH terminus of mouse RhBG and mouse RhCG weregenerated and shown to be specific to these proteins. Theseantibodies were then used to detect the specific cellular locations ofRhBG and RhCG in the normal mouse kidney.! s% ]6 e3 j# x2 c3 Z4 U/ d3 e- P
+ b0 D6 c3 X' Z
METHODS
q- \, h; o( }' n8 Q3 A
& O( z! e) M1 T1 ^Antibodies# F9 m* b( }" F. y/ A" B4 `
7 B# y: a2 Q: ~$ A) [" O3 D
Synthetic peptides corresponding to a hydrophilic cytoplasmicregion near the COOH terminus of RhBG and RhCG were synthesized, purified, and coupled to keyhole limpit hemocyanin through a cysteine link by using standard techniques (Interdisciplinary Center for Biotechnology Research, University of Florida College of Medicine, Gainesville, FL). The sequences used were TETQRPLRGGESDTRA for RhBG and EEVNTVYIPEDLAHK for RhCG. Rabbit polyclonal antibodies werethen generated by using standard techniques (Cocalico Biologicals, Reamstown, PA). A mouse monoclonal anti-peptide antibody directed against RhBG was generated by using the same peptide sequence andstandard techniques (Interdisciplinary Center for Biotechnology Research).5 ]' y( V/ T* F2 z" h0 J8 v( j5 c: N v
, F7 {% _; ^- Z! ?1 FFor immunohistochemical localization of carbonic anhydrase II (CA II),we used a polyclonal antibody raised in rabbit against mouseerythrocyte CA II. This antibody has been used in previous studies toidentify mouse intercalated cells ( 36 ). These antibodies were a gift from Dr. Paul Linser (University of Florida Department ofAnatomy and Cell Biology and Whitney Marine Laboratory, Gainesville, FL), have been characterized previously ( 16, 20 ), and have been used previously to identify mouse intercalated cells( 36 ). Separate rabbit anti-pendrin antibodies were kindlysupplied by Dr. Søren Nielsen (University of Aarhus, Aarhus, Denmark)and by Dr. Ines Royaux (National Institutes of Health, Bethesda, MD) and were used at a dilution of 1:1,000 unless otherwise detailed. Bothhave been characterized in detail previously ( 17, 34, 41 ).Rabbit anti-thiazide-sensitive transporter (TSC) antibodies weresupplied by Dr. Stephen Hebert (Yale University), have been characterized previously ( 33 ), and were used at a dilutionof 1:1,000. Antibodies against AE1 were kindly provided by Dr. Philip S. Low (Purdue University, West Lafayette, IN), have been characterized previously ( 36, 37 ), and were used at a dilution of 1:400.; a5 O/ E9 S' w
5 a- X+ p' N, n% d5 W7 a# ~. u5 {Membrane Protein Preparation) |5 g' R/ K3 |; B7 y# z
# R2 f. @; Y) |. e5 w
Normal BALB/c mice were obtained from Harlan Sprague Dawley(Indianapolis, IN) and were maintained on a normal mouse diet and adlibitum water intake until the day of study. The animals wereanesthetized intraperitoneally with pentobarbital sodium (50 mg/kg bodywt), and the kidneys were rinsed by in vivo cardiac perfusion with PBS(pH 7.4), rapidly removed, and stored frozen at 70°C until used.Tissues were then homogenized with a Tissue Tearor homogenizer (BioSpecProducts) and then diluted in buffer B (250 mM sucrose, 10 mM Tris buffer, and 1 mM EDTA, pH 7.4) containing PMSF. The sample wasthen centrifuged at 1,000 g for 10 min at 4°C. Thesupernatant was removed and centrifuged at 10,000 g for 20 min at 4°C. The supernatant was decanted, and the 10,000 g centrifugation was repeated twice more. The collected supernatants werethen centrifuged at 100,000 g for 1 h at 4°C. Thepellet was resuspended in 500 µl buffer B and gentlyhomogenized in a Dounce homogenizer. An aliquot was obtained forprotein determination by using a Lowry assay, and the remainder wasstored frozen at 70°C until used.
6 \$ m9 J1 a7 `* _# Q9 N
p: h( |2 U6 zImmunoblotting Procedure' s6 X% Q0 ^! b$ e: g
9 V# v9 s3 U! D5 K c1 o# h
Five micrograms of the renal membrane protein wereelectrophoresed on 10% PAGE ReadyGel (Bio-Rad, Hercules, CA). Gelswere then transferred electrophoretically to nitrocellulose membranes, blocked with 5 g/dl nonfat dry milk, and incubated for 1 h with primary antibody diluted 1:1,000 in Blotto buffer (50 mM Tris, 150 mMNaCl, 5 mM Na 2 EDTA, and 0.05% Tween-20, pH 7.5) with 5 g/dl nonfat dry milk. After washing, membranes were exposed to secondary antibody (goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase; Promega, Madison, WI) at adilution of 1:5,000. Sites of antibody-antigen reaction were visualizedby using enhanced chemiluminescence (SuperSignal West Pico Substrate,Pierce, Rockford, IL) and a Kodak Image Station 440CF digital imaging system.2 p, J- h% ~. N6 y {
; ]: L) W) ~' r& z' O; S7 ~0 n0 a
Tissue Preparation for Immunohistochemical Localization of RhBGand RhCG
6 {6 E. Z* h6 r- Z6 W2 }% l
: E: F# ]. ]) P8 K/ ~2 b- IFemale BALB/c mice weighing 17-20 g ( n = 6)were anesthetized with pentobarbital sodium (10-30 mg/kg ip). Thekidneys were preserved by in vivo cardiac perfusion with PBS (pH 7.4)followed by periodate-lysine-2% paraformaldehyde (PLP)( 30 ) and then cut transversely into several 2- to4-mm-thick slices and immersed overnight at 4°C in the same fixative.Samples of kidney from each animal were embedded in polyester wax(polyethylene glycol 400 distearate, Polysciences, Warrington, PA), and5-µm-thick sections were cut and mounted on gelatin-coated glass slides.
6 E. g# w9 P1 I# j7 [3 w5 O$ f, ]/ v! f
Immunohistochemistry) a0 U6 k) j$ v; W/ w7 d' ]$ \
8 l' ], h6 B" Z& w
Immunolocalization of RhBG and RhCG was accomplished by usingimmunoperoxidase procedures and a commercially available kit (Vectastain Elite, Vector Laboratories, Burlingame, CA). The sections were dewaxed in ethanol and rehydrated, rinsed in PBS, treated for 15 min with 5% normal goat serum (Vector Laboratories) in PBS, and thenincubated at 4°C overnight with either the anti-RhBG or anti-RhCGantibody, diluted 1:10,000 in PBS. The sections were then washed inPBS, and endogenous peroxidase activity was blocked by incubating thesections in 0.3% H 2 O 2 for 30 min. The sections were washed in PBS and incubated for 30 min with biotinylated goatanti-rabbit IgG secondary antibody (Vector Laboratories) diluted 1:200in PBS and again washed with PBS. The sections were treated for 30 minwith the avidin-biotin complex reagent, rinsed with PBS, and thenexposed to diaminobenzidine. The sections were washed in distilledwater, and in some experiments the sections were counterstained withhematoxylin. The sections were then dehydrated with xylene, mountedwith Permount (Fisher Scientific, Fair Lawn, NJ), and observed by light microscopy.- z ]$ z) a% @4 N
1 O- v, s E: I0 K/ o8 T& L2 ~
In some experiments localizing RhCG, the methods described above weremodified by inclusion of an antigen retrieval step. In theseexperiments, after the slides were dewaxed in ethanol and rehydrated,they were microwaved at medium power in 0.1 M sodium citrate and 0.1 Mcitric acid, pH 6.0, for 10 min. The slides were then rinsed in PBSbefore blocking with normal goat serum and incubation with the primaryantibody at a 1:8,000 dilution.7 I1 H7 v) k$ U# |0 H% ~# j
& l+ Y. w# _7 \! X! f
Colocalization of RhBG and RhCG with CA II Immunoreactivity
; _. M: W$ P. u- |% q+ @! L/ w3 P7 v# _/ p4 m7 W" t1 h) r. n6 B. x
Colocalization was accomplished by using sequentialimmunoperoxidase procedures and Vectastain Elite. Five-micrometersections were dewaxed in ethanol, rehydrated, and then rinsed in PBS.Endogenous peroxidase activity was blocked by incubation of thesections in 0.3% H 2 O 2 for 30 min. The sectionswere rinsed in PBS, treated for 20 min with 5% goat serum in PBS, andthen incubated at 4°C overnight with either the anti-RhBG or theanti-RhCG antibody diluted 1:10,000 in PBS. The sections were washed inPBS for 1 min, in 0.1% SDS in PBS for 10 min, and then again in PBSfor 1 min. The sections were then incubated for 30 min with thebiotinylated goat anti-rabbit IgG secondary antibody diluted 1:200 inPBS and then washed with PBS. The sections were treated for 30 min with the avidin-biotin complex reagent, rinsed with PBS, and then exposed todiaminobenzidine. The sections were washed in glass-distilled water andthen in PBS and incubated in 0.3% H 2 O 2 for 30 min. The sections were again washed in PBS and incubated for 20 minwith 5% normal serum in PBS. The sections were treated for 60 min with the anti-CA II antibody diluted 1:1,600 or 1:2,000 in PBS, washed inPBS, and incubated with the biotinylated anti-rabbit secondary antibody. The sections were washed with PBS, incubated with the avidin-biotin complex reagent, and washed with PBS. For detection of CAII immunoreactivity, Vector SG (Vector Laboratories) was used as thechromogen to produce a blue label. This label was easilydistinguishable from the brown label produced by the diaminobenzidine used for detection of RhBG or RhCG immunoreactivity. The sections werewashed with glass-distilled water, dehydrated with xylene, mounted withPermount, and observed by light microscopy.
6 j2 A6 R) ?1 n2 y* `& e8 {9 U9 y/ x) N
Colocalization of RhBG with TSC, RhCG, AE1, and Pendrin1 o6 u- G3 N3 ?4 r6 h0 G" {
8 O: y: w: s6 [% O
Immunofluorescent localization and confocal laser scanningmicroscopy were used in some studies to colocalize RhBG with RhCG, TSC,AE1, or pendrin. In these studies, we used our mouse monoclonal RhBGantibody; the other primary antibodies were rabbit polyclonal antibodies, thereby facilitating separate identification of their localization by using species-specific secondary antibodies. Briefly, the sections were dewaxed with ethanol, rehydrated, rinsed in PBS, andthen treated for 30 min with 50 mM NH 4 Cl. They were then rinsed in PBS, blocked for 20 min with 5% normal goat serum (Vector Laboratories) in PBS, and incubated at 4°C overnight with primary antibody, or antibodies, diluted in PBS. The sections were then washedin PBS and incubated for 30 min with a fluorescently tagged species-specific secondary antibody [either FITC-labeled goat anti-rabbit IgG, 1:50 dilution (Sigma), or AF488-labeled goat anti-rabbit IgG, 1:100 dilution, and tetramethylrhodamineisothiocyanate-labeled goat anti-mouse IgG, 1:50 dilution (Sigma)]diluted in PBS. The sections were then rinsed with PBS and mounted byusing Fluoromount (Southern Biotechnology Associates, Birmingham, AL).We then visualized the tissue by using either an Axiovert 100 M laserscanning confocal microscope (Carl Zeiss, Thornwood, NY) with LSM 510 Software, version 2.8 (Carl Zeiss), or an MRC-1024 laser scanningconfocal microscope and LaserSharp software (Bio-Rad Laboratories).; b, ^4 d$ D6 a: g
& s" Q; j0 s) g1 \) WColocalization of RhBG with Pendrin
r# h; i1 S+ c# Z0 a% _9 K& I) o s: N5 A4 y: }/ i
We performed double labeling both on wax-embedded mouse kidneyand on 1-µm sections embedded in Epon 812, as summarized below.7 b0 h3 a# c0 M7 K
# c3 l* e! k8 R% G1 F
Double labeling on wax-embedded mouse kidney. Slices of PLP-fixed kidney tissue were dehydrated and embedded inpolyester wax, and sections were cut and mounted on gelatin-coated glass slides. The sections were dewaxed with xylene and ethanol, andafter rinsing in tap water, sections were treated with methanolic H 2 O 2 for 30 min. Before incubation with theprimary antibody, the sections were permeabilized by incubation for 15 min in 0.5% Triton X-100 in PBS, subsequently blocked with normal goatserum, diluted 1:10 in PBS, for 1 h, and then incubated overnightat 4°C with rabbit antiserum against pendrin (kindly provided by Dr.Søren Nielsen) diluted 1:3,000 in PBS. The sections were rinsed in PBSand incubated for 2 h in peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Sections were then incubated with the peroxidase-substrate solution, a mixtureof 0.05% 3,3'-diaminobenzidine and 0.01%H 2 O 2, for 5 min at room temperature. Afterbeing rinsed with Tris · HCl buffer, the sectionswere treated with the same protocol for the second antiserum,polyclonal antibodies directed against RhBG, diluted 1:1,000 in PBS,except for the use of a Vector SG kit (Vector Laboratories) as thechromogen to produce a gray-blue label, which is easily distinguishedfrom the brown label produced by 3,3'-diaminobenzidine in the firstimmunolocalization procedure for pendrin. The sections were rinsed intap water and examined with light microscopy.
5 J, q" u0 j# ]# q Y ~, u, F3 H* w8 n
Double labeling on 1-µm Epon 812-embedded sections. Sections of PLP-fixed tissue were cut transversely on a Vibratome at athickness of 50 µm and processed for immunohistochemistry by using anindirect immunoperoxidase method as described in detail in a previousstudy ( 17 ). All sections were washed with 50 mM NH 4 Cl in PBS three times for 15 min. Before incubation withthe primary antibodies, all tissue sections were incubated for 3 h with PBS containing 1% bovine serum albumin, 0.05% saponin, and 0.2%gelatin ( solution A ). The tissue sections were thenincubated overnight at 4°C with rabbit antiserum against pendrin,1:3,000 dilution, in PBS plus 1% bovine serum albumin ( solutionB ). After several washes with solution A, the tissuesections were incubated for 2 h in peroxidase-conjugated goatanti-rabbit Fab fragment (Jackson ImmunoResearch Laboratories), diluted1:100 in solution B. The tissues were then rinsed, first in solution A and subsequently in 0.05 M Tris (hydroxymethyl)aminomethane (Tris) buffer, pH 7.6. For the detection of horseradishperoxidase, the sections were incubated in 0.1% 3,3'-diaminobenzidinein 0.05 M Tris buffer for 5 min, after whichH 2 O 2 was added to a final concentration of0.01% and the incubation was continued for 10 min. After washing with0.05 M Tris buffer three times, the sections were dehydrated in agraded series of ethanol and embedded in Epon 812. From the flat-embedded 50-µm-thick sections, areas from the cortex were excised and glued onto empty blocks of Epon 812, and consecutive 1.5-µm sections were cut for postembedding immunolabeling. The sections were treated for 5 min with a saturated solution of sodium hydroxide to remove the resin. After three brief rinses in absolute ethanol, the sections were hydrated with graded ethanol and rinsed intap water. The sections were rinsed with PBS, incubated in normal goatserum for 30 min, and subsequently incubated overnight with anti-RhBGantibody (1:3,000) at 4°C. The sections were rinsed with PES andincubated for 2 h in peroxidase-conjugated donkey anti-rabbit IgG,Fab fragment, and washed again with PBS. For detection of RhBGantibody, Vector SG was used as the chromogen, and the sections werewashed with distilled water, dehydrated with graded ethanol and xylene,mounted in Canada balsam, and examined by light microscopy.' C% N5 l; I/ D% F/ f/ n
/ U* K* b2 H2 bControls. Q% s4 |. B7 C- j' j
# N9 c+ i+ P6 p; X- r5 oFor controls in each of the immunolocalization procedures,preimmune serum diluted at the same concentration as the primary antibody in PBS or PBS only was substituted for the primary antibody. In other experiments, the polyclonal RhBG and RhCG antibodies wereincubated overnight at 4°C with an excess of the antigenic peptide,and the antigen/antibody mixture was substituted for the primaryantibody. For colocalization procedures, in each experiment controlsincluded substitution of buffer only for the first primary antibody,the second primary antibody, and both primary antibodies.
# T# I N6 K4 n D% n% H' U) Z5 m
RESULTS
! d( X, g$ n' V1 N7 _# u7 d% {- P1 g; `4 p6 W" v1 ?; [
Immunoblot Analyses+ h3 @; T# S& Z' R( N
$ [+ J, D* Z" i6 |& H W/ P
Immunoblot analyses of membrane protein isolated from mouse kidneyby using both the anti-RhBG and the anti-RhCG antibodies demonstratedspecific immunoreactive proteins at the appropriate molecularmasses. The anti-RhBG polyclonal and monoclonal antibodies recognized a native protein of ~52 kDa (Fig. 1, A and B, respectively), consistentwith the reported molecular mass of 50-55 kDa for RhBG ( 23 ). The monoclonal anti-RhBG antibodies alsorecognized a faint band at ~40 kDa. This may represent thenonglycosylated form of RhBG ( 23 ). Importantly, allimmunoreactivity was blocked by coincubation with the immunizingpeptide with both the polyclonal and the monoclonal RhBG antibodies.The anti-RhCG antibodies recognized a protein of ~58 kDa (Fig. 1 C ), consistent with the reported molecular mass of ~58kDa for RhCG ( 22 ). An additional band at ~46 kDa was observed; coincubation with the immunizing peptide did not alterrecognition of this band, suggesting that it represents nonspecificcross-reactivity. With all three antibodies, the broad band seen on theimmunoblot is consistent with the known glycosylation of these proteins( 22, 23 ). Control immunoblots in which the primaryantibody was substituted with the respective preimmune sera werenegative (not shown). These results identify that both RhBG and RhCGprotein are expressed in normal mouse kidney.
3 W, ~8 p- b; S7 i4 @, a2 C: n' L4 W+ R. y" m
Fig. 1. A : polyclonal anti-Rh B-glycoprotein (RhBG) antibodies.An immunoblot utilizing 5 µg of a mouse renal membrane vesiclepreparation is probed with rabbit polyclonal anti-RhBG antibodies. B : monoclonal anti-RhBG antibody. An immunoblot utilizing 5 µg of a mouse renal membrane vesicle preparation is probed with amouse monoclonal anti-RhBG antibody. C : polyclonal anti-RhC-glycoprotein (RhCG) antibodies. An immunoblot utilizing 5 µg of amouse renal membrane vesicle is probed with rabbit polyclonal anti-RhCGantibody. Arrows, appropriate band.
; m0 X, Y- k. c6 n/ l) u
4 @# `; R4 L! d! s7 _6 NImmunolocalization of RhBG: E/ M; [' y5 N% N7 h1 x3 |; f
6 h/ ~/ @3 t6 v# M0 }: H! TIntense basolateral RhBG immunoreactivity was observed inepithelial cells in the connecting segment (CNT) and in the collecting duct throughout the cortex and outer medulla and extending into theinner medulla (cortex, Figs. 2 and 3; medulla, Fig. 4 ). Proximal tubules and thickascending limbs of Henle's loop were negative. Parallel studies with asecond polyclonal anti-RhBG antiserum and with a monoclonal anti-RhBGantibody yielded identical results (not shown). In sections in whichthe primary antibody was substituted with either preimmune serum orbuffer only, no immunoreactivity was observed (Fig. 2 f ).Similarly, when we incubated the primary antibody with the immunizingpeptide, we observed no immunoreactivity (Fig. 5, A and B ).
! H5 v/ f1 l$ r; X. G! D3 i5 z
5 v2 U/ O; h1 I) iFig. 2. Light micrographs of mouse kidney cortex labeled for RhBG by usingimmunohistochemistry; blue is due to hematoxylin counterstain. Themajority of cells in the cortical collecting ducts (CCDs) andconnecting segments (CNTs) throughout the cortex exhibit intensebasolateral immunostaining for RhBG. Proximal tubules are negative andglomeruli are faintly stained. a and b :low-magnification images of the renal cortex. a : continuousintense basolateral immunolabeling in a branched profile of the CNT(marked c in the lumen). A transition from a strongly labeled CNT tothe distal convoluted tubule (DCT), where only sporadic cells arestrongly labeled, is also evident and is illustrated at highermagnification in c (*). b : heterogeneous RhBGlocalization observed in the CCD (*). Although the great majority ofcells in the CCD exhibited strong basolateral immunostaining for RhBG,occasional cells are negative. These are illustrated at highermagnification in d (arrows). e : similar labelingin the initial collecting duct. The majority of cells were stronglypositive for basolateral RhBG, whereas occasional cells were negative(arrow). Rare cells were observed that appeared to have weakimmunostaining in the apical region of the cell (arrowhead). f : low-magnification image of negative control, in whichpreimmune serum was substituted for the primary antibody.
+ B0 ^. q% q9 m) M+ q/ S% m4 @4 S4 F0 s
Fig. 3. Light micrographs of mouse kidney cortex labeled for both RhBG(brown) and carbonic anhydrase II (CA II; blue); hematoxylincounterstain was omitted. a : multiple CNTs with continuousintense basolateral immunolabel for RhBG, which labels bothintercalated cells, identified by intense label for CA II, and CNTcells, which are negative for CA II. One profile that contains atransition from the CNT to the DCT is illustrated (*), demonstratingthe decrease in intensity of RhBG immunolabel in the majority of cellsin the DCT and strong RhBG label in intercalated cells. A portion ofthe CNT (c) is illustrated at higher magnification in c.Although the majority of intercalated cells in the CNT had discretebasolateral label only (arrows), rare cells were observed that appearedto have immunolabel apical to the cell nucleus (arrowhead). b : heterogeneous RhBG immunolabeling observed in the CCD,colocalized with CA II. Although CA II is present in both intercalatedcells and principal cells in the mouse CCD, cells that are intenselylabeled for CA II can be identified as intercalated cells. Some cellsin the CCD that had not only the characteristic shape of intercalatedcells but also intense label for CA II, were negative for RhBG(arrowhead, and illustrated at higher magnification in d ).Other intercalated cells (arrow, and illustrated at highermagnification in e ) had basolateral RhBG immunostaining thatwas more intense than that in the principal cells.
: O$ ^4 E0 t6 x' E$ h, ?6 x2 C n8 S6 y6 e* L( F
Fig. 4. Light micrographs of mouse renal medulla immunolabeled for RhBG andeither counterstained with hematoxylin ( a, b, and d ) or colabeled for CA II ( c ). In the outerstripe of the outer medullary collecting duct (OMCDo) ( A )and the inner stripe of the OMCD (OMCDi) ( b ), only asubpopulation of cells have intense basolateral immunolabel for RhBG.The majority of cells in these segments were negative for RhBG. c : colocalization of RhBG and CA II in the OMCDo,demonstrating that the RhBG-positive cells in the OMCD are also CA IIpositive and thus are intercalated cells; blue indicates CA IIimmunoreactivity. d : immunolocalization of RhBG in the innermedulla demonstrated strong basolateral labeling in a small minority ofcells in the inner medullary collecting duct (arrows), which isconsistent with the distribution of intercalated cells in thissegment.
9 }! Y" J: K! h1 p% h- |. k% _! j- V5 F2 ^3 R. J7 j% Z
Fig. 5. Prevention of RhBG and RhCG immunolabeling by coincubation withimmunizing peptide. Mouse kidney sections were examined for RhBGimmunoreactivity by using either primary antibody alone ( A )or after coincubation with the immunizing peptide ( B ).Coincubation with the immunizing peptide completely blockedimmunolabeling. Similarly, mouse kidneys were examined for RhCGimmunoreactivity by using primary antibody alone ( C ) orafter coincubation with the immunizing peptide ( D ).Coincubation with the immunizing peptide completed blocked RhCGimmunolabeling.
5 Q# y) I+ \1 W5 J' B5 b3 S
- [; l, t5 w" AIn the CNT, nearly all cells exhibited strong basolateral RhBGimmunoreactivity (Fig. 2 a ). Rarely, cells were observed that had diffuse apical label in addition to basolateral label (Fig. 3 c ). Occasionally, tubule profiles were seen that appearedto include transitions from the CNT to the distal convoluted tubule (DCT) (Fig. 2, a and c ). In these, thebasolateral label for RhBG disappeared entirely in the DCT.5 @7 ]& Q+ x, v- e( p5 a7 p' a) O
$ B2 M4 X. u5 a; a% rIn the initial collecting tubule (ICT) and in the CCD, most, but notall, cells expressed basolateral RhBG immunoreactivity. The greatmajority of cells in the ICT and CCD exhibited strong basolateralimmunoreactivity; within this group, a small population had moreintense immunoreactivity (Fig. 2, b, d, and e ). The cells with more intense basolateral immunoreactivityoften exhibited a profile that bulged into the tubule lumen, suggestingthat they were intercalated cells rather than principal cells. A smallminority of cells had no detectable RhBG immunoreactivity (Fig. 2, d and e ). Rarely, cells that had no detectablebasolateral label appeared to have weak apical immunoreactivity (Fig. 2 e ).
- G' a7 S; ~+ B7 D; p6 O
7 B) z# x. |7 g* ]7 }6 t, dIn the medulla, basolateral RhBG immunoreactivity was present only in asubpopulation of cells in the collecting duct; no other structures inthe medulla were positive for RhBG (Fig. 4 ). In both the outer and theinner stripe of the outer medullary collecting duct (OMCDo and OMCDi,respectively), the RhBG-positive cells represented a minority of thetotal cells. The incidence of positive cells in the inner medullarycollecting duct (IMCD) was less than in the OMCD and diminished in themore distal portions of the IMCD, until they disappeared entirely atthe papillary tip. In both the OMCD and the IMCD, RhBG-positive cellsfrequently had a rounded outline and bulging apical surface. Throughoutthe medullary collecting duct, the distribution of the positive cells and their morphology suggested they were intercalated cells.
( ~& i- |8 Z3 s& {9 |5 W0 r( L. }, j8 ^
Colocalization of RhBG and TSC/ \) H. u3 I7 b `1 t+ U- A* N
d6 c c* f4 z. O7 U. q4 q- n7 [
The DCT is a distinct portion of the nephron. To determine whetherRhBG is expressed in the DCT, we examined immunofluorescent colocalization of RhBG with TSC, the thiazide-sensitive Na-Cl cotransporter. TSC is expressed solely in the apical region of DCTcells ( 33 ). Using immunofluorescence, cells that expressed apical TSC did not express RhBG, and cells that expressed basolateral RhBG did not express apical TSC (Fig. 6 ).We observed several tubules with abrupt transitions from TSC-positiveto RhBG-positive cells (Fig. 6 B ). These results show thatRhBG immunoreactivity is not identifiable in the DCT by usingimmunofluorescence microscopy.
$ y e6 C# Y2 N7 I( D6 E" E- X& N
: K4 M3 q3 K! v% aFig. 6. Colocalization of thiazide-sensitive Na-Cl cotransporter(TSC) and RhBG by using immunofluorescent labeling and confocal laserscanning microscopy. TSC immunoreactivity (green) labels the apicalmembrane of DCT cells. Tubule cells that expressed apical TSC did notexpress RhBG immunoreactivity (red), and those with RhBGimmunoreactivity did not express TSC immunoreactivity ( A ).Occasionally, tubule segments with abrupt transition from TSC-positivecells to RhBG-positive cells were observed ( B ), identifyinga transition from the DCT to the CNT. These studies, as do all confocalmicroscopy colocalization studies in this report, utilize a mousemonoclonal anti-RhBG antibody.5 e( o# Y7 K }7 m. K- S/ P
, A+ h, r) s# ]% ZColocalization of RhBG with CA II
7 M# E8 z& O2 z3 ^6 E( o, Q7 ?8 s8 |' @9 P! I+ _9 F' e
The absence of basolateral RhBG immunoreactivity in apopulation of ICT and CCD cells suggested that the expression of RhBG protein is cell type specific. To begin identifying the types of cellsthat were positive or negative for basolateral immunoreactivity, wecolocalized RhBG with CA II. CA II is highly expressed by mouse CCDintercalated cells, although low expression levels are also detectablein mouse CCD principal cells ( 3 ).
( L4 E; A K% o) f1 {+ n: M) J0 r* T9 Z# W+ h0 z( u% \( B+ k
In the CNT, essentially all cells exhibited basolateral RhBGimmunoreactivity. Colocalization with CA II clearly demonstrated thatthe RhBG-positive cells included both CNT cells, or principal cells,which are CA II negative, and intercalated cells, which are strongly CAII positive (Fig. 3, a and c ). Furthermore, the rare cells that appeared to have diffuse apical RhBG labeling inaddition to the basolateral label were CA II positive and thus wereintercalated cells (Fig. 3 c )., G: M [* N5 R- R8 M c" D
3 v8 |- ?+ v: F- D: CIn the CCD, the distinction between intercalated cells and principalcells was less clear, because mouse CCD principal cells express CA IIbut at a much lower level than intercalated cells ( 3 ).However, cells that were strongly positive for CA II were easilyidentifiable, and within this group there were cells that wereintensely positive for basolateral RhBG and other cells that werenegative for RhBG (Fig. 2, b, d, and e ). The majority of CCD cells were weakly positive for CAII; thus they were principal cells and expressed basolateral RhBGimmunoreactivity that was less intense than that observed in theRhBG-positive intercalated cells. Thus all CCD principal cellsand a subset of intercalated cells exhibit basolateral RhBG immunoreactivity.
/ |( D, e( w. p+ X' g& G% t" h7 I: R, b& I
In the medullary collecting duct, CA II immunoreactivity is presentonly in intercalated cells. In the OMCD, all CA II-positive cellsexhibited intense basolateral RhBG immunoreactivity (Fig. 4 c ). Furthermore, CA II-negative cells did not expressidentifiable RhBG immunoreactivity. The results of CA II and RhBGcolocalization in the IMCD were similar to that in the OMCD. All CAII-positive cells were also RhBG positive. Thus basolateral RhBGimmunoreactivity is present in OMCD and IMCD intercalated cells and notin OMCD principal cells or in IMCD cells.
, J. E2 ?2 d o" p; z% G9 I' s
& l; v: X" [! _7 fColocalization of RhBG with AE1
3 B! B: [9 L; @$ }+ y6 M7 L. A, s" B8 d7 c0 F
To identify the specific intercalated cell subtypes that exhibitRhBG immunoreactivity, we first colocalized RhBG with AE1. AE1 ispresent in the basolateral plasma membrane of the intercalated cells inthe medulla and the type A intercalated cells in the cortex ( 1, 36, 38 ).7 g& ?8 Z5 q6 ], t
. x1 j W; `, E3 X( w; D K
In RhBG and AE1 colocalization studies, CCD, OMCD, and IMCD cellswith basolateral AE1 immunoreactivity coexpressed basolateral RhBGimmunoreactivity (Fig. 7 ). OMCD and IMCDcells that were AE1 negative were also RhBG negative. Thus CCD A-typeintercalated cells and intercalated cells in the OMCD and IMCD expressbasolateral RhBG immunoreactivity.
9 Q4 T; k {" a. e% a" I
/ M- t9 \( `# B h7 R! c! \Fig. 7. Colocalization of AE1 and RhBG in the CCD. BasolateralAE1 immunoreactivity (green) identifies type A intercalated cells( A ) and is shown with white arrows. A single erythrocyte ina peritubular capillary (red arrow) is also identified. BasolateralRhBG immunoreactivity is present in the majority of CCD cells( B ) but is absent in occasional cells (arrowhead).Colocalization of AE1 with RhBG ( C, yellow) identifies thatall CCD type A cells express strong basolateral RhBG immunoreactivity.Cells without identifiable basolateral RhBG immunoreactivity do notexpress basolateral AE1 immunoreactivity.0 }" R3 r/ Q* {0 _9 `
1 t; i9 O% \9 g# n! c; t) w+ C* ~ OColocalization of RhBG with Pendrin1 X( ?0 | I# ?& }: {5 T% R# i' Y
# H3 o3 S7 t* V3 i$ V, U% e" W
Pendrin is a recently identified apicalCl /HCO 3 − exchanger expressed in theapical membrane of both the B-type intercalated cell (B cell) and thenon-A-non-B intercalated cell ( 17, 41 ). We next examinedwhether pendrin-positive intercalated cells express RhBG immunoreactivity.2 Z& O7 m2 \* l0 c8 c
4 G( \, p9 B; }! d- m6 [% {2 H9 B7 c
The colocalization of RhBG with pendrin differed in the CCD, CNT,and ICT. In the CCD, cells that expressed apical pendrin immunoreactivity did not express basolateral RhBG immunoreactivity (Fig. 8, A and B ).Moreover, CCD cells that lacked basolateral RhBG immunoreactivityexpressed apical pendrin immunoreactivity. Occasionally, there appearedto be colocalization of apical RhBG immunoreactivity with apicalpendrin immunoreactivity. In contrast, pendrin-positive cells in theCNT almost always expressed basolateral RhBG immunoreactivity. The ICTappeared to have both pendrin-positive, RhBG-positive cells andpendrin-positive, RhBG-negative cells. Identical results were obtainedwith the two different pendrin antibodies. Thus, in general,pendrin-positive CCD cells do not express identifiable basolateral RhBGimmunoreactivity, CNT pendrin-positive cells do express basolateralRhBG immunoreactivity, and ICT pendrin-positive cells are both RhBGpositive and RhBG negative.2 @# p& g+ Z+ ?4 o% q, A
1 g; x9 B1 C* E7 U+ X5 w; d6 z
Fig. 8. Colocalization of pendrin and RhBG in the CCD and CNT. CCD cellswith apical pendrin immunoreactivity ( A, green; B, brown) do not express identifiable basolateral RhBGimmunoreactivity ( A, red; B, blue). In the CNT,almost all cells with apical pendrin immunoreactivity expressbasolateral RhBG immunoreactivity ( C and D ). A and C utilize the monoclonal anti-RhBG antibodydescribed in this study and an anti-pendrin antibody previouslycharacterized ( 34, 41 ). B and D utilize the polyclonal anti-RhBG antibody described in this study andan anti-pendrin antibody supplied by Dr. Søren Nielsen (University ofAarhus, Aarhus, Denmark) and also previously characterized( 17 ). Identical results were obtained with the 2 differentsets of anti-RhBG and anti-pendrin antibodies.
3 z3 W2 { L1 G0 K% a; W3 K8 b1 ^
8 `9 M7 v" z. S5 Q% G' J( ]- uLocalization of RhCG
! Y3 C; a8 y* _# P6 F$ E5 x t
Next, we examined the localization of RhCG protein, a secondputative ammonium transporter expressed in the kidney. RhCGimmunoreactivity was present in epithelial cells in CNT and collectingduct segments throughout the cortex, outer medulla, and inner medulla(Fig. 9 ).Coincubation of the primary antibody with the immunizing peptide completely prevented immunoreactivity (Fig. 5, C and D ). However, in contrast to RhBG, RhCG immunoreactivity wasapical rather than primarily basolateral. Identical results wereobtained by using a second anti-RhCG antibody developed in ourlaboratory (data not shown). The labeling pattern was similar insections subjected to antigen retrieval and those where antigenretrieval was omitted. However, the sections subjected to antigenretrieval exhibited more distinct labeling., S9 Y3 p- e7 C0 T I# p' f$ }
- S& S7 l5 z* j, HFig. 9. Light micrographs of mouse kidney labeled for RhCGby using immunohistochemistry after antigen retrieval. The segmentaldistribution of RhCG was similar to that of RhBG in that it was presentin the CNT and in collecting ducts throughout the kidney. Proximaltubules and distal tubules were negative. However, in contrast to RhBG,the cellular distribution of RhCG immunolabel was apical rather thanbasolateral. a : low-magnification image of RhCG immunolabelin mouse kidney cortex illustrating the intense apical localization inthe CNT (c) and CCD (*). b : higher magnification of CNT in a. The apical label in the CNT was intense in most cells,although some cells appeared to have broader apical labeling (arrows),whereas other cells had weaker apical labeling (arrowhead). c : higher magnification image of CCD illustrated in a. In the CCD, a minority of cells exhibited broad, intenseapical immunolabel for RhCG (arrows), whereas the remainder of cellshad narrower and weaker apical immunolabel (arrowheads). d  uter stripe of mouse outer medulla. Apical immunolabel was present invirtually all cells in the mouse OMCDo. However, as in the CCD,heterogeneity in the intensity of label was present, with a minority ofcells exhibiting intense, broad apical immunoreactivity (arrows) andthe majority of cells labeled with only a thin apical band(arrowheads). e : inner stripe of mouse outer medulla.Intense apical immunolabel was present in a minority of cells in theOMCDi (arrows), whereas the majority of cells in thissegment were negative. f : inner medulla. Only occasionalcells exhibited apical immunolabel (arrow). g :low-magnification image of negative control, in which preimmune serumwas substituted for the primary antibody.
' H. }: v( j* k1 |2 V9 H5 d) i( Q9 H# a1 j0 p; S5 ^9 r* w) X
In the cortex, virtually all cells in the CNT, ICT, and CCD exhibitedapical labeling (Fig. 9, a-c ). The apical labeling of cells in the CNT was more intense and homogeneous than in the CCD(compare Fig. 9, b and c ). In the CCD, themajority of cells exhibited weak apical immunolabeling, whereas a smallpopulation of cells had intense apical immunolabeling (Fig. 9 c ). The intensely labeled cells frequently had a rounded,bulging apical surface, suggesting that they were intercalated cells.RhCG immunoreactivity was not observed in proximal tubule cells orthick ascending limb cells, even in experiments in which higherconcentrations of the primary antibody were used.) a4 Z, _$ o% f) m: g
) z( T* U2 e0 |- b; O
The OMCDo expressed RhCG immunoreactivity that was apical and presentin virtually all cells (Fig. 9 d ). The pattern was similar tothat observed in the CCD, with the majority of cells having weak apicallabeling and a minority exhibiting intense apical labeling. However, inthe OMCDi, the majority of cells were negative for the RhCGimmunolabel, although a minority had intense apical labeling (Fig. 9 e ). In the IMCD, only a small minority of cells exhibitedapical immunolabeling (Fig. 9 f ).7 b5 E! z0 u" s/ M" p7 j
+ O2 T" l6 J* W t: o1 z
Colocalization of RhCG with RhBG! s9 V8 ]( F: z$ s; d6 J: X& s! p
. i% N% O) j w. U2 e, W. z
The convoluted tubule cells in which RhCG is expressed could bethe same cells in which RhBG is expressed, i.e., the CNT, or it couldbe the DCT or both the DCT and the CNT. To differentiate thesepossibilities, we examined colocalization of RhBG and RhCG immunoreactivity. In all cases, convoluted tubule cells either expressed apical RhCG and basolateral RhBG or expressed neither (Fig. 10 ). Thus RhCG is expressed in the sameconvoluted tubule cells as RhBG, i.e., CNT cells, and not in DCT cells." }% d' {' R7 s: l
4 R! p4 g) i+ d: A0 S* `Fig. 10. Colocalization of RhBG and RhCG. CNT cells with apicalRhCG immunoreactivity (green) expressed basolateral RhBGimmunoreactivity (red) and vice versa.+ T2 F0 ~ {" Y7 x$ V2 q s/ o
# y) b' D# u3 W' U2 {% d# pColocalization of RhCG and CA II
& g" }6 ]1 k# o8 s$ {# S
0 T5 F- s9 q, j( f) mIn the CNT, colocalization of RhCG with CA II demonstrated thatessentially all CA II-negative cells, that is, CNT cells, expressedapical RhCG immunoreactivity (Fig. 11, a and c ). The majority of CAII-positive cells expressed intense RhCG apical immunoreactivity thatwas much greater than that observed in CA II-negative cells. However, afew CA II-positive cells exhibited only faint or no immunoreactivity(Fig. 11 c ).0 D1 E3 K) F: p# j- @7 q
& n( M$ z# k4 }4 B4 Q+ U' u
Fig. 11. Light micrographs of mouse kidney labeled for both RhCG(brown) and CA II (blue). a and b :low-magnification images of the renal cortex illustratingimmunolocalization in the CNT and CCD, respectively. c :higher magnification image of the CNT in A. CNT cells(arrowheads), which are negative for CA II, exhibit apical immunolabelfor RhCG, as do the majority of intercalated cells (black arrows),which are identified by CA II immunolabel. Occasional intercalatedcells (gray arrow) exhibited only faint label for RhCG. d :higher magnification of the CCD illustrated in b,demonstrating that intercalated cells that were intensely labeled forCA II exhibited strong apical RhCG immunolabel (arrows). Principalcells (arrowheads) also had apical RhCG immunoreactivity, but the labelwas less prominent. e and f : outer stripe of theouter medulla. e : low-magnification image demonstratinglabeling in the OMCDo. f : higher magnification imagedemonstrating that in the OMCDo, both intercalated cells, which were CAII positive (arrows), and principal cells (arrowheads) had apical RhCGimmunoreactivity, although the label was more prominent in intercalatedcells. g : inner stripe of the outer medulla. h :inner medulla. In the OMCDi and the inner medullary collecting duct,only intercalated cells were RhCG positive (arrows).6 E8 B; X9 g( m" M: u7 ]: ^
1 E8 ^4 ?- g. K7 hIn the CCD, although the distinction between principal cells andintercalated cells was not as clear as in the CNT, cells identified asintercalated cells by strong CA II immunoreactivity exhibited intenseapical RhCG immunoreactivity (Fig. 11, b and d ).The majority of cells had weaker CA II immunoreactivity and a thinapical band of RhCG immunoreactivity. Thus principal cells exhibitedless abundant apical RhCG compared with intercalated cells.- M- j1 d0 Z: C% K
7 p! _# e# p( AAlthough RhCG immunoreactivity was present in all cells in the OMCDo,the immunolabel was more intense in CA II-positive cells than in CAII-negative cells (Fig. 11, e and f ). Thus theOMCDo intercalated cell appears to contain more apical RhCG than the OMCDo principal cell." y; _* ^$ O* d$ g8 O6 R, G5 T9 {
8 s( n. h- _5 f. O4 MIn the OMCDi and the IMCD, RhCG immunoreactivity was present only in CAII-positive cells (Fig. 11, g and h ). Thus inthese segments, RhCG is present in intercalated cells and is notdetectable in the OMCDi principal cell or in the IMCD cell.
* O' r& w# c% j, y- ?5 M2 m
7 B9 N' {0 C# b5 _' m# WThus apical RhCG immunoreactivity is present in virtually all cells inthe CNT, ICT, CCD, and OMCDo and in intercalated cells in the OMCDi andIMCD. In the cortical segments and the OMCDo, intercalated cells weremore intensely labeled than principal cells.
; Y& o6 \! @, k6 {7 o. W; J5 V; T
: \1 \4 I: j: e* K) s! _3 \DISCUSSION
1 t" N [8 W: X+ @6 ?; n, g# Y: i/ E" C6 H
The present studies are the first to determine the specificcellular location of the putative ammonium transporter family proteinsRhBG and RhCG in the mouse kidney. Our findings demonstrate that theputative ammonium transporters RhBG and RhCG are widely distributedthroughout the collecting duct and CNT. However, the intensity of theimmunoreactivity and the prevalence of positive cells are markedlygreater in the CNT and the CCD than in the medullary collecting ductsegments. We find that in the CCD and CNT virtually all epithelial cellsubtypes express both basolateral RhBG and apical RhCG, including CNTcells, principal cells, and type A and non-A-non-B intercalated cellsbut, interestingly, not type B intercalated cells.
; i+ Q8 c {0 _5 D9 t) ]( c3 G+ u8 g m$ @% U/ D7 r
In medullary collecting duct segments, the coexistence of basolateralRhBG and apical RhCG persists in the intercalated cells, which aresimilar in function to type A intercalated cells in the CCD. However,expression of these transporters is largely absent in the majority celltypes, principal cells, and IMCD cells and is limited to weak apicalRhCG immunoreactivity in OMCDo principal cells.* T: h, Z% Q0 g1 G$ f
7 @- E/ ^ ^0 e5 FThe coexistence and polarized expression of these transporters inmultiple epithelial cell subtypes in collecting duct segments and themarked axial heterogeneity with respect to both the intensity ofimmunoreactivity and the prevalence of labeled cells, particularly among principal cells, suggest that RhBG and RhCG mediate specific andimportant roles in the kidney.$ a- `- k' s" t
4 t$ K: l( X3 _6 q6 S* F+ H5 qThe cellular localization of RhBG protein in the present study adds tothe reported localization of RhBG. In situ hybridization studiessuggested that renal convoluted tubules express RhBG mRNA ( 23 ). Whether these were proximal convoluted tubules,DCTs, or CNTs was not determined. The present studies identify no RhBG immunoreactivity in proximal convoluted tubule cells, even in experiments in which we used higher concentrations of the RhBG antibodythan was necessary to produce intense immunoreactivity in the CNT andcollecting duct. Thus our findings of intense RhBG immunoreactivity inthe CNT but not in proximal tubules or DCT suggest that the convolutedtubules that were positive for RhBG mRNA in the in situ hybridizationstudies ( 23 ) represent the CNT. Our observation that RhBGimmunoreactivity is substantially less in the CCD than in the CNTsuggests that RhBG protein expression is less in the CCD than in theCNT. Thus CCD RhBG mRNA expression may also be lower, potentially belowthe level of detection by using in situ hybridization. This mightexplain the failure to detect RhBG mRNA expression by in situhybridization in a previous study ( 23 ).$ h3 i- d& u1 N: \2 X, {* W3 o
% \- \- h4 }2 l& A _ z/ GThe present studies demonstrate that a subpopulation of CCDintercalated cells do not express identifiable basolateral RhBG immunoreactivity. Colocalization of RhBG with AE1 demonstrates that thetype A intercalated cell in the CCD, as well as intercalated cellsthroughout the medullary collecting duct, express basolateral RhBG.Colocalization with pendrin, an apical anion exchanger present in typeB and non-A-non-B intercalated cells ( 17, 41 ), revealed two patterns of RhBG localization in pendrin-positive cells. In theCCD, pendrin-positive cells were negative for RhBG, whereas in the CNTpendrin-positive cells typically exhibited basolateral RhBGimmunoreactivity. In the mouse CNT, non-A-non-B intercalated cells areprevalent and type B intercalated cells are rare ( 36 ). Thus the pendrin-positive, RhBG-positive cells observed in the CNTlikely are non-A-non-B intercalated cells. In the CCD, the identity ofnon-A intercalated cells is not as clear. Some investigators havereported that in the CCD, type B intercalated cells occur frequentlyand non-A-non-B intercalated cells are rare or absent ( 36 ), whereas other studies have reported that non-A-non-Bintercalated cells occur as frequently as type B intercalated cells( 15 ). Thus the pendrin-positive, RhBG-negative cellsobserved in the CCD likely include type B intercalated cells. It isalso possible that non-A-non-B intercalated cells in the CCD, unlikethose in the CNT, do not express RhBG. Nevertheless, our findingsindicate that the CNT non-A-non-B intercalated cell and type A cellsthroughout the medullary collecting duct express basolateral RhBGimmunoreactivity, whereas the CCD type B cell does not express it atdetectable levels.+ U3 [; K4 N6 c$ ~% f% ^
! N Y6 o& n& ]9 mThe cellular localization of RhCG protein that we observed in thisstudy is both consistent with and adds to a previous report examiningRhCG mRNA expression in the mouse kidney ( 22 ). Using insitu mRNA hybridization, it was suggested that RhCG mRNA was expressedby collecting duct cells ( 22 ). The immunohistochemical findings in the present study are consistent with this observation andextend these findings in important ways. First, the present studyestablishes that RhCG protein is present in the CNT and the collectingduct. In addition, the present study demonstrates that RhCG is locatedat the apical membrane of mouse renal cells in which it is expressed.We have observed similar findings in the rat (Verlander and Weiner,unpublished observations).$ J1 a3 H) O6 V3 {
* B( Z% x4 e+ p# k3 K9 y; h
Although our findings of polarized expression of RhBG and RhCG in manyspecific epithelial cells in the CNT and collecting duct suggest theseproteins have functional importance, their potential roles in thekidney presently must be surmised from evidence regarding the functionsof related proteins in other cell types. In 1994, separate independentgroups reported the cloning and expression of ammonium transportersfrom the yeast Saccharomyces cerevisiae, Mep1( 29 ), and from plants, Amt1 ( 32 ). Both Mep1and Amt1 encode proteins of ~54 kDa with 9-12 transmembrane domains that transport both ammonium (NH 4 + ) and[ 14 C]methylammonium, a radiolabeled ammonium analog( 29, 32 ). Further studies have identified multipleadditional members of the ammonium transporter family: Mep2 and Mep3 inyeast ( 28 ) and Amt1;2, Amt1;3 and Amt2 in plants( 10, 35 ). In the last few years, members of the ammoniumtransporter family have been identified in essentially all organisms,from bacteria to the slime mold, Dicytostelium discoideum,birds, and mammals ( 2, 12, 13, 39 ).% T/ Z7 p7 s5 @
$ C: M/ i) z, N, O; p
A fundamental characteristic of yeast and plant members of this proteinfamily is ammonium transport ( 26, 27, 29 ). RhAG, anothermammalian member of this transport family ( 26 ), transports the ammonium analog methylammonium ( 42 ). Similarly, apreliminary report suggests that mouse RhCG, when expressed in the Xenopus laevis oocyte, mediates electrogenic acid loading inthe presence, but not the absence, of extracellular ammonia, consistentwith RhCG functioning as an electrogenic NH 4 + transporter ( 31 ). Thus it is possible that RhBG or RhCG,or both, mediate transepithelial ammonium transport by the CNT and collecting duct, which are critically important sites for total ammoniasecretion ( 4, 18 ).
- q8 ]! m1 ]) T8 N
) c2 k/ ?" n. e) ^6 mAnother possible function of these proteins is to function as ammonia"sensors." In the mammalian collecting duct, ammonia regulatesseveral aspects of collecting duct ion transport. For example, ammoniastimulates collecting duct net proton secretion ( 6, 9, 19, 40 ) and potassium reabsorption ( 11 ).Simultaneously, ammonia inhibits CCD unidirectional bicarbonate andpotassium secretion and sodium reabsorption ( 7, 11 ). Themechanisms through which ammonia alters ion transport are notcompletely identified. However, the effects of ammonia on CCD acid-basetransport can be dissociated from ammonia transport( 7-9 ). Thus ammonia may act through a cellular sensorto regulate collecting duct ion transport. Evidence from otherorganisms suggests that ammonium transporters related to RhBG and RhCGcan serve as ammonia sensors and regulate cell function. Specifically,in yeast, the ammonium transporter Mep2 regulates the cellular responseto nutrient depletion through mechanisms that are independent ofammonium transport ( 25 ). Thus it is possible that RhBG orRhCG may function as such a sensor and may regulate function in any ofthe specific epithelial cell types where they are present.
' P8 r! l2 S1 V5 u4 `! `' g% u7 j( t) K
In summary, these studies are the first to identify the cellularlocalization of the ammonium transporter family of proteins RhBG andRhCG in the mouse kidney. RhBG and RhCG protein exhibit axialheterogeneity and polarized expression in many specific epithelial celltypes in the mouse CNT and collecting duct, consistent with animportant role for these proteins in either ammonium transport orsignaling throughout these segments.$ q$ A! v0 C; G' {0 ~0 o: l
X# L" l! `7 s6 {2 N; {
NOTE ADDED IN PROOF j: g; F0 }' X
" I0 [" x9 d' w+ z, u+ V8 V
A report describing a similar localization of RhCG in the ratkidney ( 5 ) was published while this manuscript was under review.! N! Y& C' ]9 {4 t) X
9 I2 a. K9 V/ V0 d. n" a
ACKNOWLEDGEMENTS
8 y& p# g& u; T( N/ d. W
2 d+ B* w& `, H* X) Q% e( K0 VThe authors thank Gina Cowsert and Lee Ann Day for secretarialsupport and Melissa A. Lewis and Lauren DeWitt of the University ofFlorida College of Medicine Electron Microscopy Core Facility whoperformed the majority of the immunohistochemical experiments. We alsothank Dr. Shen-Ling Xia of the North Florida/South Georgia VeteransHealth System confocal microscopy core facility and Timothy Vaught ofthe University of Florida Brain Institute for assistance with theconfocal microscopy.% {0 W5 l1 C2 e7 L4 B6 B# `+ k
【参考文献】: \( b' e; C7 ^& F2 J$ P3 W
1. Alper, SL,Natale J,Gluck S,Lodish HF,andBrown D. Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H -ATPase. Proc Natl Acad Sci USA 86:5429-5433,1989 .7 r& c- W' ^# x, `
- M! V0 j2 R. n. \* Z7 l8 Y. e/ G% o6 p1 [* v! c) G; y
$ r# b- T2 K a6 I
2. Avent, ND. A new chapter in Rh research: Rh proteins are ammonium transporters. Trends Mol Med 7:94-96,2001 .
$ T1 g9 U/ O# ?; `' o
- i2 K* r. |" l+ t; z2 i- `; x: U P+ f
+ _; t5 z, M5 V3 @0 U
3. Dobyan, DC,Magill LS,Friedman PA,Hebert SC,andBulger RE. Carbonic anhydrase histochemistry in rabbit and mouse kidneys. Anat Rec 204:185-197,1982 .& a J8 ~+ }/ e' B% R
( y; n0 K1 y/ S5 N3 s6 O
' I0 b8 ^6 s; b4 r( B6 y+ M0 J" X
9 N- ?$ t6 d4 [1 J" V; ^4. DuBose, TD, Jr,Good DW,Hamm LL,andWall SM. Ammonium transport in the kidney: new physiological concepts and their clinical implications. J Am Soc Nephrol 1:1193-1203,1991 .* r9 K8 P$ g- H* Q ~
* n8 Z$ m2 ]' f
/ M$ e; G! [ p1 d; v2 F1 }: W. v K) V
5. Eladari, D,Cheval L,Quentin F,Bertrand O,Mouro I,Cherif-Zahar B,Cartron JP,Paillard M,Doucet A,andChambrey R. Expression of RhCG, a new putative NH 3 /NH 4 + transporter, along the rat nephron. J Am Soc Nephrol 13:1999-2008,2002 .
_8 a) m: S: X: o) Z. O! t0 M7 Z% f5 g3 K% m9 u- S0 Q
" V- |. m9 d0 _9 ^
/ y; r5 M$ X( G& _6. Flessner, MF,Wall SM,andKnepper MA. Ammonium and bicarbonate transport in rat outer medullary collecting ducts. Am J Physiol Renal Fluid Electrolyte Physiol 262:F1-F7,1992 .
+ K @- j, G; \' b6 s
^7 R8 H: L; m6 i" U$ y) G1 N3 D6 O
6 Q3 R& v( Y2 f5 v7 {, B& w
4 U+ ^0 g4 r6 n0 h1 L7. Frank, AE,andWeiner ID. Effects of ammonia on acid-base transport by the B-type intercalated cell. J Am Soc Nephrol 12:1607-1614,2001 .# ]- Q3 J. P' a( B
$ h2 o& t3 @+ R6 @
% ^3 ?, x2 w3 R" O: P6 h7 u+ Z* P0 Z$ i- E2 t' t) }
8. Frank, AE,Wingo CS,Andrews PM,Ageloff S,Knepper MA,andWeiner ID. Mechanism by which ammonia stimulates cortical collecting duct bicarbonate reabsorption. Am J Physiol Renal Physiol 282:F1120-F1128,2002 .
; ~& p9 h1 o7 p0 C4 Z* Q4 B ]
8 T4 z. f3 k( m5 `! m9 p/ }% ^- L3 D* n+ s. E1 b
% x2 X/ `7 l( B ?+ S! W; C
9. Frank, AE,Wingo CS,andWeiner ID. Effects of ammonia on bicarbonate transport in the cortical collecting duct. Am J Physiol Renal Physiol 278:F219-F226,2000 .+ o) n4 I$ v4 n0 Z- Z0 i& ^# T
* T. h/ m# B. @. z3 u2 g- ?
$ }$ B4 x8 i8 A& A- H% }& Z% b
1 g6 e' s g; d% V+ {
10. Gazzarrini, S,Lejay L,Gojon A,Ninnemann O,Frommer WB,andvon Wiren N. Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell 11:937-948,1999 .# h. E; C6 i8 ]/ i
, z7 `( P+ E# D7 r+ b
$ B" Y: A- @1 m4 o8 l# v
2 Q. u4 p2 z' n) V' q; D" g11. Hamm, LL,Gillespie C,andKlahr S. NH 4 Cl inhibition of transport in the rabbit cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 248:F631-F637,1985 .& y5 d+ `3 ?# @' _, l( W8 }0 R2 Q
3 B7 I5 U' r! i
, K4 I( U; p: `" Q @( {
8 t1 h; I1 e( X5 T12. Heitman, J,andAgre P. A new face of the Rhesus antigen. Nat Genet 26:258-259,2000 .' D0 N( A5 V2 X( h+ v
5 j1 M3 H" v: \) I
3 \9 `: V R& q) e' X8 U" q0 c! Q( G5 E- H: `/ ^% i
13. Howitt, SM,andUdvardi MK. Structure, function and regulation of ammonium transporters in plants. Biochim Biophys Acta 1465:152-170,2000 .4 I2 A8 o& g* e) R
I# }6 J% }, U
& G5 B7 y' U! G/ o- l1 R" N
5 e6 R! r9 }. k& m9 n3 K& ~14. Huang, CH,andLiu PZ. New insights into the Rh superfamily of genes and proteins in erythroid cells and nonerythroid tissues. Blood Cells Mol Dis 27:90-101,2001 .& J+ ?- F' B9 z. b' V, Y
) b* K( N1 m8 W7 o( M
6 f7 s4 f! i& k! C# ^5 N# H: T) L* K6 Y& y" Q3 V6 a; u" ]
15. Kim, J,Kim YH,Cha JH,Tisher CC,andMadsen KM. Intercalated cell subtypes in connecting tubule and cortical collecting duct of rat and mouse. J Am Soc Nephrol 10:1-12,1999 .
- a# m6 F, C9 K, M2 O0 ?" Z& }. {4 e! e
1 s6 y: b# l' \+ s" ]
4 o% M" o5 r8 X: N u16. Kim, J,Tisher CC,Linser PJ,andMadsen KM. Ultrastructural localization of carbonic anhydrase II in subpopulations of intercalated cells of the rat kidney. J Am Soc Nephrol 1:245-256,1990 .
7 O& Y8 l: D( n6 k" }
2 U" V$ v/ [( q: d/ U7 ^
' Y1 T. \" k7 B. A" }4 e7 u) g- v2 C, Z& M
17. Kim, YH,Kwon TH,Frische S,Kim J,Tisher CC,Madsen KM,andNielsen S. Immunohistochemical and immunoelectron microscopic localization of pendrin in intercalated cell subtypes in rat and mouse kidney. Am J Physiol Renal Physiol 283:F744-F754,2002 .5 s& U( r1 h8 F2 q# i) e" X4 _
) f4 p" Z, [* G0 V) O
" o! P) T2 {1 y$ j0 n9 [9 E3 z
6 V$ [: Q% O3 C2 W5 [ V+ V9 b' o4 y18. Knepper, MA. NH 4 + transport in the kidney. Kidney Int 40:S95-S102,1991." a7 m" m( R; _$ K3 g& D
+ d; I( v5 |: f9 A5 d2 ^6 C% c) V' l" v+ l
! a5 R* H. [6 B b$ f' f
19. Knepper, MA,Good DW,andBurg MB. Ammonia and bicarbonate transport by rat cortical collecting ducts perfused in vitro. Am J Physiol Renal Fluid Electrolyte Physiol 249:F870-F877,1985 .
/ @( d7 h" w: k8 v
. M, {" j( O. k3 q6 ]
$ y, [% F* T3 a4 H
6 E1 b6 z: G$ _% m9 N8 j( X20. Linser, PJ,Sorrentino M,andMoscona AA. Cellular compartmentalization of carbonic anhydrase-C and glutamine synthetase in developing and mature mouse neural retina. Brain Res 315:65-71,1984 .
7 h% {2 Q& F9 V/ m4 u
|' f6 ?) k5 q$ X% w$ c* Y- P& e" X! q9 C8 t, j
; a* y+ T1 T9 B6 Z7 u6 [
21. Liu, L,Dunn ST,Christakos S,Hanson-Painton O,andBourdeau JE. Calbindin-D28k gene expression in the developing mouse kidney. Kidney Int 44:322-330,1993 .
& Y+ R& M% r$ z1 O* n
/ R" I$ S4 q8 h) l% L: Q1 M5 e
; O( p* s1 J1 I
* k: h8 U4 I# g9 W/ |22. Liu, Z,Chen Y,Mo R,Cc Hui Cheng JF,Mohandas N,andHuang CH. Characterization of human RhCG and mouse Rhcg as novel nonerythroid Rh glycoprotein homologues predominantly expressed in kidney and testis. J Biol Chem 275:25641-25651,2000 .
# K7 g h) @7 [
7 H, o+ k' x/ [+ g" h+ E4 S/ [4 X8 `: k- }1 L/ o& Y6 z
+ F ]2 t/ O4 T
23. Liu, Z,Peng J,Mo R,Hui C,andHuang CH. Rh type B glycoprotein is a new member of the Rh superfamily and a putative ammonia transporter in mammals. J Biol Chem 276:1424-1433,2001 ." q0 K! w1 ]' v$ u. `) h9 a2 y/ c
5 M0 D5 K, I4 Z- P1 s
0 c# w* E/ M( W0 d E
. {1 w8 N3 W2 A+ E- v24. Lorenz, MC,andHeitman J. Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains. Genetics 150:1443-1457,1998 .% v+ v) ]; F9 `1 H, z1 P: Q
! }! y$ F& b* ]3 u2 y, A Q6 n. Q' w9 i% Y7 E F
# ~& L/ Q. x5 q
25. Lorenz, MC,andHeitman J. The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae. EMBO J 17:1236-1247,1998 .: Y1 M2 n' J% A1 N( P- b' O
) ^2 ~& ?% K- `) G/ K& r
5 S; n# s X9 r: x# `1 m# O+ `
" D. M! q" ]. _! s. W! Y
26. Marini, AM,Matassi G,Raynal V,Andre B,Cartron JP,andCherif-Zahar B. The human Rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast. Nat Genet 26:341-344,2000 .- s' P$ m: m* D0 I' b9 p4 A
6 _& s# j. B( _' Z( X, u
1 Q% t- X) @8 j$ E) `/ b- P* }) t
/ i0 }1 ]* ^) R; T( w6 ]0 @27. Marini, AM,Soussi-Boudekou S,Vissers S,andAndre B. A family of ammonium transporters in Saccharomyces cerevisiae. Mol Cell Biol 17:4282-4293,1997 .
B! p' L; |. f2 L
- A8 p5 P/ y, w& B; G1 c Y8 S! L- H9 w+ b1 R* v4 E
* m/ |1 Y! ^; G9 n! F5 x/ p28. Marini, AM,Vissers S,andAndre B. Ammonium transport in Saccharomyces cerevisiae. Yeast 11:425,1995 .* z' g ^" f$ B! D* R
$ h+ }4 N+ M$ l
2 f0 `1 [& @9 @$ H8 {2 _8 U2 R) V
29. Marini, AM,Vissers S,Urrestarazu A,andAndre B. Cloning and expression of the MEP1 gene encoding an ammonium transporter in Saccharomyces cerevisiae. EMBO J 13:3456-3463,1994 .
8 @ O. j8 Q% m" u& @) R+ a; l' |3 k2 {8 i# ]3 v' z0 O7 M8 T
0 v' F% x2 a5 F1 F
! `* l' O3 O4 V3 Y5 L2 s" D30. McLean, IW,andNakane PK. Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J Histochem Cytochem 22:1077-1083,1974 .
( I, x: c$ |" E$ N
. \2 B5 j- L1 B7 C: m9 `4 b w( l1 I; L3 m! x4 a' S2 }
0 b( x- I5 o, ~' z! p
31. Nakhoul, NL,Palmer SA,Abdulnour-Nakhoul S,Hering-Smith K,andHamm LL. Ammonium transport in oocytes expressing Rhcg (Abstract). FASEB J 16:A53,2002. s# i9 g; |) G. a5 F8 Z( T
! y1 s$ s/ `) V3 B# R" p9 q
# U' ]4 d5 ?3 R; a3 A
O7 g% x8 q6 S6 z+ w9 |32. Ninnemann, O,Jauniaux JC,andFrommer WB. Identification of a high affinity NH 4 + transporter from plants. EMBO J 13:3464-3471,1994 .
4 d1 ?% z1 u. f8 P3 x; y8 a: Q. U, s- o c
. W) F# U! a& Q
3 W L+ O' e! k$ C9 R" t/ f6 s33. Plotkin, MD,Kaplan MR,Verlander JW,Lee WS,Brown D,Poch E,Gullans SR,andHebert SC. Localization of the thiazide sensitive Na-Cl cotransporter, rTSC1 in the rat kidney. Kidney Int 50:174-183,1996 .
# {! W/ O$ [8 w6 B' ^) o9 N/ X4 I w2 _9 ?$ I& P
% ~0 r1 W) C+ P! ]
" ^6 B* |9 x0 U/ R. M8 h
34. 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 .
, p7 V+ P( } D6 j% u
% }; W4 b- l# F9 @- y4 M% T6 Q* L: q- V2 z) z/ k% P
' G8 }9 u( ?9 M" C1 v
35. Sohlenkamp, C,Shelden M,Howitt S,andUdvardi M. Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants. FEBS Lett 467:273-278,2000 .
# k/ T* O8 G; R7 R$ e7 _& Z$ M4 N
* \' i) R. r- y2 Z4 d' Y1 ~" F8 n; M
' g, h6 B+ ^ O2 ?3 {* J: v36. Teng-umnuay, P,Verlander JW,Yuan W,Tisher CC,andMadsen KM. Identification of distinct subpopulations of intercalated cells in the mouse collecting duct. J Am Soc Nephrol 7:260-274,1996 .8 v# o( K( l H @8 f( W# E
+ b/ G% V% t/ C! i) T8 `; O- v" I6 r9 b$ L8 W3 l. A
J4 m0 I- Q# ^
37. 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 .5 d& [! D: D7 O/ z j7 I" D4 n
- H9 i3 B3 p; p. I4 o0 a7 B' `
; b* E, I2 h+ f9 u9 Y. i/ ]! {3 ?# t% }) F
38. Verlander, JW,Madsen KM,andTisher CC. Axial distribution of band 3-positive intercalated cells in the collecting duct of control and ammonium chloride-loaded rabbits. Kidney Int 57:S137-S147,1996.# S6 \2 r y1 p
3 ~, \% ]9 X& Y/ v* r
V6 O; b, T7 \" u5 u9 [" l, Z# `% @
' S1 i3 b! ^. a) B% X7 t1 z39. Von Wiren, N,Gazzarrini S,Gojon A,andFrommer WB. The molecular physiology of ammonium uptake and retrieval. Curr Opin Plant Biol 3:254-261,2000 .
# i1 @ h* B' e' Q1 v$ Q1 U6 k7 o S0 H% `( `
8 F9 Z) T6 }7 F; \/ ^2 W" F& u/ u1 U
40. Wall, SM. NH 4 + augments net acid-secretion by a ouabain-sensitive mechanism in isolated-perfused inner medullary collecting ducts. Am J Physiol Renal Fluid Electrolyte Physiol 270:F432-F439,1996 .
8 m, | v% {, }+ y" ^( n. V$ U
9 A+ [8 x" q; \0 b% a
; F' i8 A8 U5 q, M) ?1 D
1 J, }$ f! N+ y41. Wall, SM,Hassell KA,Royaux IE,Green ED,Chang JY,Shipley GL,andVerlander JW. Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol 284:F229-F241,2003 . First published August 27, 2002; 10.1152/ajprenal.00147.2002.
; n0 f6 G N; `
; x3 p& y- c5 ~& k/ x
a1 s* L1 ]! j( t- j$ o+ d/ z6 b% g6 l, n7 ^5 O# n
42. Westhoff, CM,Ferreri-Jacobia M,Mak DD,andFoskett JK. Identification of the erythrocyte Rh-blood group glycoprotein as a mammalian ammonium transporter. J Biol Chem 277:12499-12502,2002 . |
|
发表于 2009-4-21 13:32
