Plasticity of mouse renal collecting duct in response to potassium depletion

doi: 10.1152/physiolgenomics.00055.200419:61-73, 2004. First published 6 July 2004;Physiol. Genomics

Jean-Marc Elalouf and Alain DoucetLydie Cheval, Jean Paul Duong Van Huyen, Patrick Bruneval, Jean-Marc Verbavatz,potassium depletionPlasticity of mouse renal collecting duct in response to

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Plasticity of mouse renal collecting duct in response to potassium depletion

Lydie Cheval,1 Jean Paul Duong Van Huyen,2 Patrick Bruneval,2

Jean-Marc Verbavatz,3 Jean-Marc Elalouf,3 and Alain Doucet1

1Laboratoire de Physiologie et Genomique Renales, Unite Mixte de Recherche Centre National de la RechercheScientifique/UPMC 7134, and2Groupe d’Anatomo-Pathologie, INSERM U430, IFR 58, Institut des Cordeliers,75270 Paris; and3Departement de Biologie Joliot Curie, CEA Saclay, 91191 Gif sur Yvette, France

Submitted 4 March 2004; accepted in final form 30 June 2004

Cheval, Lydie, Jean Paul Duong Van Huyen, Patrick Bruneval,Jean-Marc Verbavatz, Jean-Marc Elalouf, and Alain Doucet.Plasticity of mouse renal collecting duct in response to potassiumdepletion. Physiol Genomics19: 61–73, 2004. First published July 6,2004; doi:10.1152/physiolgenomics.00055.2004.—Renal collectingducts are the main sites for regulation of whole body potassiumbalance. Changes in dietary intake of potassium induce pleiotropicadaptations of collecting duct cells, which include alterations of ionand water transport properties along with an hypertrophic response.To study the pleiotropic adaptation of the outer medullary collectingduct (OMCD) to dietary potassium depletion, we combined functionalstudies of renal function (ion, water, and acid/base handling), analysisof OMCD hypertrophy (electron microscopy) and hyperplasia (PCNAlabeling), and large scale analysis of gene expression (transcriptomeanalysis). The transcriptome of OMCD was compared in mice fedeither a normal or a potassium-depleted diet for 3 days using serialanalysis of gene expression (SAGE) adapted for downsized extracts.SAGE is based on the generation of transcript-specific tag libraries.Approximately 20,000 tags corresponding to 10,000 different molec-ular species were sequenced in each library. Among the 186 tagsdifferentially expressed (P � 0.05) between the two libraries, 120were overexpressed and 66 were downregulated. The SAGE expres-sion profile obtained in the control library was representative ofdifferent functional classes of proteins and of the two cell types(principal and �-intercalated cells) constituting the OMCD. Combinedwith gene expression analysis, results of functional and morphologicalstudies allowed us to identify candidate genes for distinct physiolog-ical processes modified by potassium depletion: sodium, potassium,and water handling, hyperplasia and hypertrophy. Finally, comparisonof mouse and human OMCD transcriptomes allowed us to address thequestion of the relevance of the mouse as a model for humanphysiology and pathophysiology.

transcriptome; serial analysis of gene expression; kidney collectingduct; hypertrophy; hyperplasia

POTASSIUM CONTROLS MANY CELL functions, the most important ofwhich is membrane potential. This control rests both on themaintenance of whole body potassium content (�3,600 mmolin a 70-kg human) and the distribution of potassium betweenintra- and extracellular compartments (�3,500 mmol and 100mmol, respectively). Homeostasia of whole body potassiumrelies on the continuous balance between dietary intake (�100mmol/day) and excretion of potassium. Kidneys are not onlyresponsible for the bulk of potassium excretion (�90%) butalso for the adaptive changes in potassium handling in responseto alterations of its dietary intake. This renal plasticity toward

potassium handling is quantitatively important, as urinary po-tassium excretion may vary over 20-fold between potassiumrestriction and potassium loading conditions.

Renal handling of potassium along the renal tubule resultsfrom complex reabsorption and secretion processes: �90% ofthe filtered load of potassium is reabsorbed between the glo-merulus and the distal convoluted tubule, whereas the down-stream collecting duct usually secretes potassium into thetubular fluid. The collecting duct is the main site of homeosta-sic adjustments of the excreted load of potassium: secretion isenhanced in case of increased dietary intake of potassium,whereas during potassium restriction, the secretion process isabolished and replaced by a reabsorption mechanism (31),which allows recovery of most of the potassium remaining inthe tubular fluid beyond the distal convoluted tubule. It isgenerally assumed that inhibition of potassium secretion duringpotassium restriction mainly originates in cortical collectingduct (CCD), whereas the potassium reabsorption mechanismprevails in the outer medullary collecting duct (OMCD) whereit would be accounted for in part by the induction of a nongastricH-K-ATPase at the apical pole of principal cells (30).

Because the transports of water and of the different solutesin the collecting duct are directly or indirectly coupled at themolecular and/or cellular levels, adaptation to dietary potas-sium restriction alters not only the transport of potassium butalso that of water and other solutes. Furthermore, functionaladaptations of the collecting duct not only affect the transportfunctions of the different cell types but may include alsochanges in the energy metabolism (to balance ATP availabilitywith active ion transport) (39), cellular hypertrophy and in-crease in apical and/or basolateral cell membrane surface(which increases the exchange surface available for transport)(26), cell proliferation (thereby increasing the number of trans-porting cells) (35, 50), or even cell transdifferentiation (whichmodifies the ratio of the different cell types that constitute thecollecting duct and thereby the proportion of cells dedicated tothe different transport processes) (1). All these adaptive re-sponses are likely mediated through a complex network ofinduction/repression of gene expression. Thus functional ad-aptations of the collecting duct appear as pleiotropic phenom-ena that include changes in the expression pattern of manydifferent genes.

The present study was designed to analyze the pleiotropicadaptation of the collecting duct to dietary potassium deple-tion. For this purpose, we compared the molecular phenotypeof collecting duct from mouse fed either a normal or a potas-sium-depleted diet. The study was restricted to the OMCDbecause of its major role in potassium conservation in potas-sium depletion and of its lesser cellular heterogeneity com-pared with the CCD. The pattern of gene expression was

Article published online before print. See web site for date of publication(http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. Doucet, UMR7134, Institut des Cordeliers, 15 rue de l’Ecole de Medecine, 75270 Pariscedex 6, France (E-mail [email protected]).

Physiol Genomics19: 61–73, 2004.First published July 6, 2004; doi:10.1152/physiolgenomics.00055.2004.

1094-8341/04 $5.00 Copyright © 2004 the American Physiological Society 61



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evaluated using the serial analysis for gene expression (SAGE)microassay compatible with microdissected OMCDs previ-ously developed in the laboratory (59).

METHODS

Animals. Experiments were carried out on two groups of maleC57BL/6J mice (8–10 wk old; Charles Rivers Breeding Laboratories).Mice were fed either a potassium-deficient diet (LK) containing 1.5meq K�/kg (SAFE, Epinay, France) for 3 days (LK-3d) or 14 days(LK-14d), or a similar diet supplemented with KCl (150 meq/kg)(control). All animals had free access to food and were allowed todrink deionized water ad libitum.

For metabolic studies, animals were housed in individual metaboliccages (Phymep, Paris, France) starting 3 days before the beginning ofthe experiments. Then, 24-h urine samples were collected undermineral oil for 2 days under the K�-supplemented diet followed by 14days on the LK diet. On the same mice, blood was sampled byretro-ocular puncture at day 2 on the regular diet and by exsanguina-tion at day 14 on the LK diet, for determination of plasma sodium,potassium, calcium, magnesium, and creatinine concentrations. Urinevolume and osmolality were gravimetrically and cryometrically de-termined, respectively. Plasma and urine concentrations of creatinine,sodium, potassium, calcium, and magnesium were determined with anautomatic analyzer (Hitachi 911; Boehringer Mannheim). Glomerularfiltration rate (GFR) was evaluated by the clearance of creatinine.

In an other group of animals, blood samples were obtained byretro-ocular puncture in control, LK-3d, and LK-14d mice. A singlepuncture was obtained from each animal. Blood pH and bicarbonateconcentration were determined with a blood gas analyzer (ABL30;Radiometer, Copenhagen, Denmark).

In a last series, kidneys were removed from control, LK-3d, andLK-14d mice for determination of fresh and dry weights.

mRNA extraction and generation of SAGE libraries. Two SAGElibraries were prepared from OMCD of control and LK-3d mice,respectively. After anesthesia (pentobarbital sodium, 140 �g/g bodywt), the left kidney was perfused with Hanks’ modified microdissec-tion solution, then with the same solution supplemented with 0.15%collagenase (Serva, Heidelberg, Germany). The kidney was slicedalong the corticopapillary axis in small pieces which were incubatedfor 20 min at 30°C in collagenase-containing (0.25%) microdissectionsolution. After rinsing, microdissection was performed at 4°C understereomicroscopic observation.

Approximately 400 and 600 OMCDs dissected from two controland two LK-3d mice were used to generate SAGE libraries. Librarieswere generated by using the SAGE adaptation for downsized extracts(SADE) method (59). The main modifications of this protocol consistsof the use of oligo(dT)25 covalently bound to magnetic beads (Dyna-beads mRNA direct kit; Dynal, Oslo, Norway) to purify poly(A)RNAs, with the use of Sau3AI as the anchoring enzyme.

Sequencing was performed on DNA minipreps by using Big Dyeterminator sequencing chemistry (Applera) and an automated se-quencer (ABI 377, Applera). Sequence files were analyzed usingSAGE2000 software (58). Tags corresponding to linker sequenceswere discarded, and those originating from duplicate ditags werecounted only once. Significant differences between the two librarieswere assessed by Monte-Carlo simulation analysis (63), with P �0.05 being considered as significant.

Identification of tags. Tags present at statistically different levels inthe two libraries were identified through matching to UniGene clustersusing the SAGEmap resource (http://www.ncbi.nlm.nih.gov/SAGE)at NCBI. The reliability of tag identification was validated by con-firming the correct location and orientation of the tag in relation to the3� most Sau3AI site, as well as the presence of a polyadenylationsignal and/or poly(A) tail. In some instances, expression of a singlegene was detected through two tags contiguous to the 3� most Sau3AIsite and to the penultimate one. This feature was exclusively observed

for tags with high occurrence, consistent with incomplete Sau3AIdigestion of the cDNA. These tags were referred to as A and B. Fortags reliably matching sequences belonging to two unrelated clusters,the two entries were recorded. Tags reliably matching �2 clusterswere referred to as multiple matches. For tags without reliableidentification, we searched whether they might correspond to neighborof identified tags. Neighbors, which were defined by the presence ofa single nucleotide difference (mutation or shift) with the original tagand thereby likely correspond to sequencing errors or genetic poly-morphism, are identified by an asterisk in the list in SupplementalTable S3 (available at the Physiological Genomics web site).1

mRNA extraction and RT-PCR analysis. The following structureswere microdissected from collagenase-treated kidneys according tomorphologic and topographic criteria: proximal convoluted andstraight tubules (PCT and PST), medullary and cortical thick ascend-ing limb of Henle’s loop (MTAL and CTAL), and CCD and OMCD.Total RNAs were extracted from pools of nephron segments (1–2 cmlength) by using a microadaptation (12) of the method of Chomczyn-ski and Sacchi. RT-PCR was carried out on total RNAs correspondingto 1 mm of kidney tubule, which corresponds to �400 cells. Reversetranscription and PCR were performed sequentially in the samereaction tube in the presence of [�-32P]dCTP (106 Bq/nmol). Theprimers (Supplemental Table S1) were selected (Oligo 4.0; Med-Probe, Oslo, Norway) from mouse sequences available from Gen-Bank. RT-PCR products were separated by electrophoresis on 2%agarose slab gels and quantitated with a PhosphorImager (MolecularDynamics, Sunnyvale, CA). The number of PCR cycles was adjustedto provide nonsaturating signals detectable by PhosphorImager butnot by ethidium bromide staining.

When the expression profile of a transcript along the nephron wasanalyzed, RNAs from the different nephron segments were processedin the same experiment, and in each experiment, the RT-PCR productquantitated by using PhosphorImager (arbitrary units) in each struc-ture was calculated as percent of the amount in the PCT. Results areexpressed as means � SE from several experiments in differentanimals.

When comparing expression levels in OMCDs from differentgroups of mice (control, LK-3d, and LK-14d), RNAs from at least onecontrol and one LK mouse were analyzed in each experiment, andvalues for the LK animal were expressed as percent of the control.Results are expressed as means � SE from several experiments indifferent animals.

Statistical analysis between groups was performed by varianceanalysis, with P � 0.05 being considered as significant.

Ultrastructure of OMCD. Kidneys from the three groups of micewere fixed by in situ perfusion with 2% glutaraldehyde in PBSfollowed by a 1-h incubation in the same solution and washed in 0.1M cacodylate buffer (pH 6.8). Tissues were postfixed for 1 h in a 1/1mixture of 2% aqueous osmium tetroxide and 3% aqueous potassiumferrocyanide. Dehydration was performed in graded ethanol bathsfollowed by Epon embedding (Sigma, St Louis, MO). We cut 90-nmsections with a Reichert ultramicrotome (Leica, Wetzlar, Germany),and we counterstained these for 2.5 min with 1% lead-citrate beforeexamination at the electron microscope (model EM400; Philips,Limeil Brevannes, France).

Proliferating cell nuclear antigen (PCNA) labeling. Kidneys fromthe three groups of mice were fixed by in situ perfusion with formalin.Double immunohistochemistry with anti-AQP-2 and anti-PCNA an-tibodies was performed using a three-step streptavidin-biotin methodwith prior antigen unmasking procedure. Deparaffinized 5-�m thickkidney sections were heated at 98°C for 30 min in TRS buffer(DAKO, Trappes, France). Endogenous peroxidase, avidin, and biotin

1The Supplementary Material for this article (Supplemental Tables S1–S3)is available online at http://physiolgenomics.physiology.org/cgi/content/full/00055.2004/DC1.

62 TRANSCRIPTOME OF KIDNEY COLLECTING DUCT IN K DEPLETION

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activities were blocked using 0.3% H2O2 and avidin and biotinblockers (DAKO), respectively. Monoclonal anti-AQP-2 1321 anti-body (kindly provided by Dr. A. Blanchard, INSERM U356) was thenincubated at 1.25 �g/ml, for 60 min, at room temperature. Secondarybiotinylated anti-mouse IgG antibody (Amersham, Les Ulis, France)was used at 1/200 for 30 min. Streptavidin-peroxidase amplificationwas then performed with the ELITE kit (Vector, Burlingame, CA)according to the manufacturer’s instructions using diaminobenzidine(DAKO) as a chromogen. After rinsing in PBS, the sections wereincubated with anti-PCNA monoclonal antibody (PC10 clone,DAKO) at 1/200 for 60 min, at room temperature. Secondary biotin-ylated anti-mouse IgG antibody (Amersham) was used at 1/200 for 30min. Streptavidin-phosphatase procedure was performed with theABC-phosphatase kit (Vector) using Vector blue chromogen (Vector).The slides were then counterstained with hematoxylin. PCNA labelingin OMCD in principal (AQP-2 positive) and intercalated (AQP-2negative) cells was determined by counting an average of 750 cellsper animal in a blinded fashion.

RESULTS

Metabolic studies. Table 1 summarizes body weights andurinary parameters at day 2 on K�-supplemented diet (control)and days 3 and 14 on LK diet, and plasma parameters on thesame mice at day 2 on K�-supplemented diet (control) and day14 on LK diet. As previously reported in rats (24), body weightdecreased in mice fed the LK diet. Plasma creatinine concen-tration (Pcreat) decreased in parallel with the body weight,suggesting that the latter was mainly due to a loss of muscularmass. Decrease in urinary excretion of creatinine (UcreatUV)in LK-14d mice was higher than the decrease in Pcreat, reveal-ing a 50% reduction in the glomerular filtration rate (GFR).

Urine flow rate (UV) remained constant during the first weekon LK diet and then decreased by 30% at day 14. This effectwas likely accounted for, at least in part, by the decrease inGFR. Decrease in urinary excretion of potassium (UKUV)markedly exceeded the decrease in the filtered load of potas-sium: UKUV decreased by 53% at day 1, 90% at day 3, andthereafter slowly decreased toward 95% at day 14. Despite thisreduction in urinary potassium excretion and in muscle mass,plasma potassium concentration only dropped by 35% after 14days on LK diet. Urinary excretion of sodium (UNaUV)decreased in parallel with GFR, and plasma sodium concen-tration was not modified after 14 days on LK diet. Excretion ofcalcium (UCaUV) also decreased in excess of GFR, but thisdid not alter significantly plasma calcium concentration. Incontrast, the marked reduction in magnesium excretion(UMgUV), which was proportional to that of potassium,induced a significant hypermagnesemia. The parallelism be-tween UKUV and UMgUV observed during potassium de-pletion was previously reported during dietary magnesiumrestriction (60), suggesting a tight coupling between renaltransport of these two ions. However, the cellular and molec-ular mechanisms of magnesium transport in the distal nephronremain as yet unknown.

Conversely to rats, which develop a metabolic alkalosis inresponse to potassium depletion (27), mice displayed a slighttendency to develop a moderate acidosis at day 14.

OMCD hypertrophy and hyperplasia. The increase in thefractional kidney weight observed after 3 days of potassiumdepletion was totally accounted for by the decrease in bodyweight, as the fresh and dry kidney weights were not differentin control and LK-3d mice (Table 2). In contrast, after 14 days,the fresh and dry kidney weights were increased by 13%,revealing a marked hypertrophy and/or hyperplasia.

Electron microscopic observation revealed that OMCD prin-cipal and intercalated cells had similar heights in control andLK-3d mice (Fig. 1, A and B), revealing the absence ofhypertrophy. Nonetheless, intercalated cells displayed an am-plification at their luminal pole, with the development ofnumerous microplicae of apical membrane and, to a lesserextent, at their serosal pole with enhanced digitation processesof the basal membrane. Ultrastructure of principal cells was notaltered significantly. In contrast, in LK-14d mice both celltypes were markedly hypertrophied and had invaded the tubu-lar lumen, which appeared as virtual (Fig. 1, C and D). Also,

Table 1. Urine and blood parameters

Control LK-3d LK-14d

Body wt, g 21.6�0.4 18.9�0.4† 16.5�0.3†§UV, ml/day 1.5�0.1 1.5�0.2 1.0�0.2*UOsm UV, �Osm/day 2,790�267 1,781�190* 2,021�140†§Pcreat, �M 44�3 33�2†Ucreat UV, �mol/day 7.4�0.3 4.5�0.4† 2.7�0.2†‡GFR, �l/min 119�7 57�5†PK, mM 5.9�0.5 3.8�0.2†UK UV, �mol/day 478�13 53�4† 24�2†§PNa, mM 141�3 139�3UNa Uv, �mol/day 201�11 183�10 102�10†§PCa, mM 2.2�0.1 2.0�0.1UCa UV, �mol/day 4.9�0.4 3.0�0.4* 0.9�0.1†§PMg, mM 1.2�0.1 2.0�0.1†UMg Uv, �mol/day 21.9�2.8 9.8�1.3* 1.7�0.2†§PpH 7.28�0.01 7.25�0.03 7.23�0.02*PHCO3, mM 21.4�0.6 19.4�1.2 19.8�1.0

Values are means � SE. Body weight and urinary parameters were followeddaily on 8 mice maintained on metabolic cages for 19 days [3 days ofaccustomization followed by 2 days on control diet and 14 days on potassium-deficient (LK) diet]. Only data corresponding to the second day on the controldiet and days 3 and 14 on LK diet are presented. In the same animals, plasmacreatinine (Pcreat), sodium (PNa), potassium (PK), calcium (Pa), and magnesium(PMg) were measured on blood sampled by retro-orbital puncture at day 2 onthe control diet and by exanguination at day 14 on LK diet. An other group ofmice was used for measurements of plasma pH and bicarbonate on bloodsampled by retro-orbital puncture on animals fed either the control diet (n �7) or the LK diet for 3 days (LK-3d, n � 7) or for 14 days (LK-14d, n � 7).UV, urine flow rate; see RESULTS for definitions of other parameters. Statisticaldifferences between groups were assessed by variance analysis. For LK-3d orLK-14d vs. control, *P � 0.05 and †P � 0.001. For LK-14d vs. LK-3d, ‡P �0.05 and §P � 0.001.

Table 2. Body and kidney weight

Control(n � 6)

LK-3d(n � 4)

LK-14d(n � 5)

Body wt, g 26.3�0.6 21.8�0.4† 22.2�0.5†Kidney fresh wt, mg 157.4�4.0 156.6�0.7 178.8�7.6*Kidney fresh wt, mg/g body wt 6.0�0.2 7.2�0.3* 8.1�0.3†Kidney dry wt, mg 39.2�0.7 38.7�2.0 44.2�1.0†Kidney dry wt, mg/g body wt 1.5�0.02 1.8�0.1* 2.0�0.03†

Values are means � SE from n animals. Body weight and kidney freshweight were determined at the time of euthanization of mice under control dietor LK diet since 3 or 14 days. Dry weight of the same kidneys was determinedafter 3 days dehydration at 60°C. Statistical differences between groups wereassessed by variance analysis. For LK-3d or LK-14d vs. control, *P � 0.05and †P � 0.005.

63TRANSCRIPTOME OF KIDNEY COLLECTING DUCT IN K DEPLETION




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the membrane alterations of intercalated cells were furtherdeveloped, especially at the basal membrane, which displayeddeep invaginations.

Hyperplasia was evaluated through quantitation of PCNA-positive cells. Principal and intercalated cells were distin-guished on the basis of the presence or absence of aquaporin 2(Fig. 2). In control mice, no PCNA-positive cell was detected,confirming the very slow proliferation rate of tubular epithelialcells in normal kidney. In contrast, OMCDs from LK-3d micedisplayed PCNA-positive cells in almost all microscopic fieldsobserved. PCNA-positive cells were more frequent amongintercalated than principal cells (Table 3). In OMCDs ofLK-14d mice, PCNA-positive cells were still detected consis-tently, but their frequency was reduced compared with LK-3dmice.

Altogether, these results indicate that adaptation to a LK dietinduces hypertrophy and hyperplasia of both principal andintercalated cells of the OMCD and that hyperplasia precedeshypertrophy, although the two phenomena overlap betweendays 3 and 14.

Transcriptome of OMCD from control mice. A total of19,698 tags were sequenced from the control mice library,corresponding to 10,521 different tags [see complete list onSupplemental Table S2 and GEO (accession numbersGSM23474 and GSM23478)]. The gene expression profile inthe OMCD of control mice was similar to that previouslyreported (59). It is characterized by the following generalfeatures (Supplemental Table S2): 1) 40% of all tags, repre-senting 75% of the molecular species, were counted only once;2) the most abundant tag represented 2% of all tags; 3) 8 ofthe 10 most represented tags are encoded by the mitochondrialgenome; and 4) the most abundant tag of nuclear origincorresponds to the mRNA encoding the water channel aqua-porin 2 (AQP-2).

The OMCD transcriptome was representative of both prin-cipal and intercalated cells as it includes specific markers ofthese two cell types; AQP-2, -3, and -4, and �-, �-, and -subunits of amiloride-sensitive epithelial sodium channel(ENaC) for principal cells, and chloride/bicarbonate exchangerkAE1, several subunits of V-type H-ATPase, Rhbg, and Rhcg

Fig. 1. Morphology of principal (P) and intercalated cells (I) in outer medullary collecting duct (OMCD) from mice fed the controldiet (A) or the LK diet for 3 days (B) or 14 days (C and D). Control mice (A) showed typical morphology of principal cells, witha smooth apical membrane (black arrowheads) and a slightly digitated basal membrane (black arrow), and intercalated cells withapical membrane displaying few microplicae (open arrowhead) and a smooth basal membrane (open arrow). After 3 days on theLK diet (B), neither the height of principal and intercalated cells nor the morphology of principal cells was altered. In contrast, themorphology of intercalated cell was already modified: these showed a larger number of apical microplicae (open arrowhead) anddeep digitations of their basal membrane (open arrow). Within 14 days of potassium depletion (C and D), both principal andintercalated cells were markedly hypertrophied: these were 3-fold taller than in control conditions, and had invaded the tubularlumen (L), which appeared as virtual. Besides their hypertrophy, principal cells displayed deeper invaginations of their basolateralmembrane, whereas the structure of their apical membrane remained basically unchanged, and intercalated cells showed moreabundant apical microplicae and a tremendous amplification of the basal membrane surface through deep and numerous digitations.Tight junctions (�) were similar in all conditions. Bars � 2 �m (A–C) or 0.5 �m (D).





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for intercalated cells (Table 4). In addition to ion transporters,the OMCD library also included tags corresponding to hor-mone receptors (vasopressin V2 and thyroid hormone recep-tors, protease-activated receptor 3), proteins involved in hor-

monal transduction and signalization (adenylyl cyclase 6, GTPbinding protein Gs, inositol trisphosphate receptor type 2,calmodulin 1), proteins involved in genetic diseases (11�-hydroxysteroid dehydrogenase 2, carbonic anhydrase 2, 56/58-kDa and 116-kDa subunits of vacuolar H-ATPase), drug tar-gets (cyclophilin and FK506-binding protein), transcriptionfactors (Sp3, Ear2, transcription repressor ring 1A), enzymesof the energy metabolism, and cytoskeleton proteins. In con-clusion, the gene expression profile obtained by SAGE isrepresentative of different functional classes of proteins and ofthe two cell types constituting the OMCD.

Differential gene expression in OMCD from control andLK-3d mice. Based on the time course of metabolic changes,we analyzed the molecular phenotype of OMCDs after 3 daysof potassium depletion, i.e., when 1) adaptation of solutetransport was almost complete, 2) cell proliferation was high,and 3) before apparent cellular hypertrophy. A total of 20,200tags corresponding to 9,833 different tags were sequencedfrom the LK-3d library (Supplemental Table S2), and thegeneral features of this library were similar to those of thecontrol one.

The number of tags differentially expressed (P � 0.05) inthe two libraries reached 186, including 120 tags overrepre-sented in the LK-3d library (Supplemental Table S3). Amongthe differentially represented tags, �60% were reliably iden-tified as single cDNAs, whereas 13% (14 tags) did not matchany GenBank sequence (Table 5). It should be noted that 11/14tags without GenBank matching sequence corresponded toneighbors of reliably identified tags. Unexpectedly, the propor-tion of reliably identified tags was much higher for tagsoverrepresented in the LK-3d library than for tags found atlower level. Over 80% of the cDNAs corresponding to differ-entially represented tags were functionally characterized. Ta-bles 6 and 7 list the tags functionally identified as nucleartranscripts that are present at higher and lower levels in theLK-3d library, respectively. Almost 50% of tags overrepre-sented in the LK-3d library encode proteins involved in energyor protein metabolism, presumably in relation with hypertro-phy and cell proliferation. Next, the most abundant classes ofoverrepresented tags correspond to proteins related to cellproliferation (17%) and to transporters (11%).

Validation of SAGE data. To validate the SAGE data,RT-PCR was performed on a selection of five tags overrepresented in the LK-3d library. Selected tags correspond toreliably and functionally identified genes: growth differentia-tion factor 15 (Gdf15), urokinase, cofilin, Slc22a4 (the organic

Fig. 2. Proliferating cell nuclear antigen (PCNA) labeling in OMCD frommice fed the control diet (A) or the LK diet for 3 days (B) or 14 days (C).Principal cells show a brown cytoplasmic labeling with anti-AQP-2 antibody,whereas intercalated cells are unstained. Anti-PCNA staining gives a strongblue signal of the nucleus. Control mice (A) showed no PCNA-positive cells inOMCD. After 3 days on the LK diet (B), foci of PCNA-positive cells werepresent mainly in intercalated cells (arrowheads) and more rarely in principalcells (inset). After 15 days on LK diet (C), the number of PCNA-positiveintercalated cells in OMCD (arrowhead) was strongly reduced.

Table 3. PCNA-positive cells in OMCD

Control(n � 9)

LK-3d(n � 9)

LK-14d(n � 8)

Principal cells 0�0 4.0�1.4* 1.4�0.7Intercalated cells 0�0 11.9�3.4† 5.0�1.6

Values are means (in percent) � SE. Proliferating cell nuclear antigen(PCNA)-positive cells in outer medullary collecting duct (OMCD) werequantitated in sections of kidney outer medulla. An average of 750 cells,corresponding to 4–6 microscopic fields (4), were counted on each mouse ofthe different groups (n � number of mice). Principal and intercalated cellswere determined as aquaporin-2-positive and -negative cells, respectively.Values statistically different were determined by ANOVA followed by theFisher PLSD test: *P � 0.005 and †P � 0.001.





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cation transporter OCTN1), and non-erythroid Rhesus proteinRhcg. RT-PCR was performed on OMCDs from control miceand mice fed the LK diet for either 3 or 14 days. As shown inFig. 3, RT-PCR confirmed the stimulation of expression ofmRNAs corresponding to four of the selected tags at day 3,whereas for the last one overexpression was revealed only after14 days of potassium depletion. After 14 days of potassiumdepletion, the overexpression averaged 2- to 6-fold, except forOCTN1 the expression of which was increased �20-fold.

Expression of OCTN1 in the collecting duct was unex-pected, since this transporter is assumed to be specific of the

proximal tubule (61). Therefore, we further investigated ex-pression of OCTN1 along the nephron and its regulation duringpotassium depletion, as well as that of other members of theorganic cation transport family, namely OCT1, 2, and 3 andOCTN2 and 3. Results in Fig. 4 confirm that the PCT and PSTare the main renal sites of expression of OCTN1. However, asignificant level of expression was also observed in the thickascending limb (both in MTAL and CTAL), whereas expres-sion level was vanishingly low, and at the limit of detection byRT-PCR, along the collecting duct. Potassium depletion didnot alter OCTN1 expression in the PCT but increased it in

Table 4. Abundance of tags for selected genes in OMCD of control and LK-3d mice and in human OMCD

Mus musculus Homo sapiens

Symbol (GenBank ID) Tag C LK Symbol (GenBank ID) Tag

Carriers, channels, and ATPasesENaC �-subunit Scnn1a (NM_011324) AGCCAAGCCA 8 12 7 SCNN1A (BC006526) ACTCCGCCTTENaC �-subunit Scnn1b (NM_011325) AGGTAAGAAG 8 5 2 SCNN1B (NM_000336) CTGGTCCTGAENaC -subunit Scnn1g (NM_011326) GTGTTTGGAG 1 3 2 SCNN1G (NM_001039) TTCCCACTTCNa-K-ATPase �1-subunit Atp1a1 (NM_144900) TTGATATTTG 8 15 4 ATP1A1 (NM_000701) TTGATATTTGNa-K-ATPase �1-subunit, long Atp1b1 (X61433) TTGTGTCAGT 28 46 23 ATP1B1 (NM_001677) TTGTATTCAGNa-K-ATPase �1-subunit, medium Atp1b1 (NM_009721) TGCCTGTCAC 12 8 1 ATP1B1 (NM_001677) TGCCCATCACNa-K-ATPase �1-subunit, short Atp1b1 (X16646) ACAAGCACAA 9 8 6 ATP1B1 (NM_001677) ACAAGCACAANa-K-ATPase -subunit Fxyd2 (NM_007503) CTTAACTTCC 4 8 3 ATP1G1 (NM_001680) TTCGCTGGACH-K-ATPase �-gastric subunit Atp4a (BC015262) CGGAAACTTG 0 0 0 ATP4A (NM_000704) CGGAAGCTTGH-K-ATPase �-gastric subunit Atp4b (NM_009724) ATTTGCCCGT 0 0 0 ATP4B (NM_000705) GTCAAGTTCCH-K-ATPase �-colonic subunit Atp12a (NM_138652) TCTGCACAAT 0 0 0 ATP12A (NM_001676) TGGGTGTATGH-ATPase 56/58-kDa subunit Atp6v1b1 (NM_134157) CCCATTCTCA 2 5 1 ATP6V1B1 (NM_001692) GACGAGTTCTH-ATPase 116-kDa subunit Atp6v0a4 (NM_080467) AAATACTAAA 3 1 1 ATP6V0A4 (NM_130841) ATGGAGGGCCROMK Kcnj1 (BC020525) TACACACACA 12 8 2 KCNJ1 (BC063190) CCCACCTGCAkAE1 Slc4a1 (NM_011403) GTGCCGCCCA 23 22 0 SLC4A1 (NM_000342) CTCCTTATCTPendrin Slc26a4 (NM_011867) TGATGGAGGC 0 0 44* SLC26A4 (NM_000441) GAGACCATCCRhBG Rhbg (NM_021375) CCCATTAGCT 7 3 3 RHBG (NM_020407) CACTGGCCCCRhCG Rhcg (NM_019799) ACTCTATTTT 26 49 1 RHCG (NM_016321) CTCTACCGACAQP-2 Aqp2 (NM_009699) GTGGCAGTGG 328 468 63 AQP2 (NM_000486) ACACACACCAAQP-3 Aqp3 (BC027400) TGAGTGGGCA 11 14 86 AQP3 (NM_004925) CGGGATGGGAAQP-4 Aqp4 (NM_009700) TGGTCAAGTT 1 0 0 AQP4 (NM_004028) AAAAATTGTGClC-K1 Clcnk1 (NM_024412) AACCCACCAG 2 2 1 CLCNKA (NM_004070) CTGGTAGGCAClC-K2 Clcnk2 (NM_019701) TCTCAGCCCA 0 0 2 CLCNKB (NM_000085) CTGGTGGGCA

Most abundant mouse OMCD tags�-Actin Actb (NM_007393) AGCAAGCAGG 46 26 105 ACTB (NM_001101) AGCAAGCAGGIntegral membrane protein 2B Itm2b (NM_008410) ACCGACCGCA 40 47 28 ITM2B (NM_021999) ACCTGGGTTTCadherin 16 Cdh16 (NM_007663) CAAGCAGCCC 36 35 6 CDH16 (NM_004062) CAGCAGGGGA11�-Hydroxysteroid

dehydrogenase type 2Hsd11b2 (NM_008289) TGACCAAGGC 32 22 8 HSD11B2 (NM_000196) CCCCAAGTGT

Insulin-like growth factor bindingprotein 7

Igfbp7 (NM_008048) ACTCTGGAGT 31 19 47 IGFBP7 (NM_001553) ACACATCAAG

Cytochrome c oxidase, subunit VIa, polypeptide 1

Cox6a1 (NM_007748) TTCCATCCCT 31 31 1 COX6A1 (NM_004373) AAAAATGGCG

Cytochrome c oxidase subunit IVisoform 1

Cox4i1 (NM_009941) GTGACTGGGT 29 25 7 COX411 (NM_001861) GGCGTGACCA

Calgizzarin S100a11 (NM_016740) CTGGTGTCCT 27 20 S100A11 (NM_005620) no Sau3AI siteRibosomal protein L11 Rp11 (NM_025919) CAAAAAGCTA 27 36 14 RPL11 (NM_000975) ATCCTTCCTGRibosomal protein S24 Rps24 (X60289) TGCGGTGACT 27 25 24 RPS24 (NM_033022) ACAGCCGAAGThymosin, �4 Tmsb4x (NM_021218) AAGTTTAAAT 26 17 34 TMSB4X (NM_021278) AAGTTTAAATATP synthase, F1 complex �-

subunitAtp5b (BC013253) TCTCCATATC 25 34 9 ATP5B (NM_001686) CTGCAGGACT

N-myc downstream regulated 1 Ndr1 (NM_010884) AGGAATCCAG 24 30 35 NDRG1 (NM_006096) GGGGCAAGAGRibosomal protein L19 Rp19 (NM_009078) AGTCTTTAAA 23 41 37 RPL19 (NM_000981) AGCCATTAAA -Actin Actg (NM_013798) TGTGCAGGGT 22 22 34 ACTG1 (BC001920) GGTGGCTCCARibosomal protein L41 Rpl41 (NM_018860.2) GCCTGGAGAA 20 43 41 RPL41 (NM_021104) ACCTCTGAGARibosomal protein S19 Rps19 (BC034506) GCTGGACAGG 20 21 10 RPS19 (NM_001022) TGGACAGAAT

Genes are identified by usual names commonly utilized by physiologists in literature. In addition, their Mouse Genome Informatics (MGI) and Human GenomeOrganisation (HUGO) symbol (for mouse and human, respectively) and GenBank identification, as well as their abundance in control (C) and LK-3d (LK) mouseOMCD libraries and in human OMCD library, are given. Data for human OMCD, normalized to 20,000 total tags, are from Chabardes-Garonne et al. (7). Theempty space for calgizzarin abundance (for Homo sapiens) indicates a transcript lacking an anchoring enzyme recognition site and therefore without SAGE tag.For pendrin, the asterisk indicates the abundance of a tag corresponding to several reliable identifications within GenBank.





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MTAL, CCD, and OMCD (1.7-, 7-, and 20-fold increases,respectively). mRNAs encoding the other members of theorganic cation transporters family, except OCT1 and OCT3,were also detected in the OMCDs, but their expression was notor only slightly modified after 14 days of potassium depletion(data not shown).

Other tags of interest. For tags with low occurrence numberin the libraries (0 or 1), a 5- to 7-fold change in abundance wasnecessary to reach the P � 0.05 significance level, whereas2-fold and 1.2-fold changes were sufficient for tags counted15 and 100 times, respectively. This means that, at the deep-ness of analysis reached in this study (20,000 tags sequenced ineach library), statistically significant differential expression oftranscript of low abundance can be determined only for tran-scripts displaying a high degree of induction. From a physio-logical point of view, it is well known that changes of muchsmaller amplitude are sufficient to induce relevant functionalalterations. Furthermore, transcriptome of the human OMCDindicated that some transporters of importance for collectingduct function are expressed at relatively low level (7). Thus wecompared the abundance in the two libraries of tags corre-sponding to transcripts encoding the main transporters ex-pressed in the collecting duct (Table 4). To compare geneexpression profiles in mouse and human OMCD, Table 4 alsolists the abundance of the cognate tags in human OMCD (7),after normalization of tag counts to 20,000 total tags perlibrary. Comparison between gene expression profiles inmouse and human OMCD was extended to all the transcriptsfrom nuclear genome corresponding to tags representing �1‰of the whole mouse OMCD transcriptome. Note that tagabundance of zero in Table 4 may indicate either 1) that thetranscript is not detectable at the depth of analysis attained inthis study (20,000 tags) or 2) that mouse OMCDs express atranscript variant with a tag different from that deduced fromthe cDNA sequence in GenBank.

DISCUSSION

Renal sodium, potassium, and water handling in LK miceand expression of related genes. Conversely to the rat, whichresponds to potassium depletion by a marked polyuria, urineflow rate decreased in LK mice. Along with this difference inurine volume, urine osmolality decreased during potassiumdepletion in rat, whereas it increased in mice. In the rat,polyuria is associated with downregulation of AQP-2 expres-

sion in the kidney cortex and medulla (37). The observedinduction of AQP-2 (Table 4) and vasopressin V2 receptor (tagabundance: ncontrol � 1; nLK-3d � 4) mRNAs expression inOMCD of LK mice may account in part for the differentbehaviors of rat and mouse. Note that expression of constitu-tive basolateral water channels AQP-3 and AQP-4 was notaltered during K depletion (Table 4).

Concerning sodium transporters, transcripts for the threesubunits of the epithelial sodium channel ENaC were detectedin the mouse OMCD transcriptome. Occurrence of their cor-responding tag was not altered consistently during potassiumdepletion (Table 4), in agreement with the maintenance of aconstant fractional excretion of sodium. In contrast, but con-sistent with previous observations in the rat OMCD (5), ex-pression of mRNAs encoding the three subunits of Na-K-ATPase was increased in OMCD of potassium-depleted mice(note that SAGE allowed us to discriminate three distincttranscripts of Na-K-ATPase �1-subunit corresponding to dis-tinct polyadenylation sites). The physiological significance ofincreased Na-K-ATPase expression in OMCD of hypokalemicanimals remains paradoxical in view of the role of Na-K-ATPase in potassium secretion in the collecting duct. Thedecreased occurrence of the tag for ROMK (Table 4), althoughnot statistically significant, may explain in part that inductionof Na-K-ATPase does not result in a parallel increase inpotassium secretion.

The main functional adaptation of collecting duct duringpotassium depletion is that instead of secreting potassium intothe lumen, it reabsorbs it to reduce the renal loss. Potassiumreabsorption along the OMCD is thought to be mediated byactive uptake of potassium across apical membrane of tubularcells via the nongastric H-K-ATPase (30), the expression ofwhich is induced during potassium depletion both in rat (38)and mouse OMCD (unpublished observation). However, thetag corresponding to the �-subunit of nongastric H-K-ATPasewas not detected in the transcriptome of LK-3d mice (Table 4)although 1) the nucleotide sequence of mouse nongastric H-K-ATPase �-subunit available in GenBank displays several re-striction sites for the anchoring enzyme (Sau3AI) that specifiesthe tag, 2) the length of this sequence is consistent with that ofthe transcript detected by Northern blot in mouse kidney (62),and 3) analysis of AU-rich element (ARE) in the 3�-untrans-lated region of mouse nongastric H-K-ATPase �-subunitmRNA allocates it to ARE Database group V transcripts (2),i.e., a class of mRNAs with normal degradation rate. In fact,assuming an mRNA abundance of nongastric H-K-ATPase�-subunit similar to that found in potassium-depleted ratOMCD (30 mRNAs per cell; Ref. 38) and a total number of300,000 transcripts per cell, the tag is expected to be countedtwice in the SAGE library (20,000 sequences analyzed) butwith a probability of only 86% (9). This example illustrates thesensitivity limit of SAGE detection for transcripts of interme-diate abundance.

Mucolipin 3 (Mcoln3), the tag abundance of which is 0 and7 in the control and LK-3d libraries, respectively, might also beinvolved in changes in transport function of OMCD duringpotassium depletion. Mcoln3 was discovered by positionalcloning in varitint-waddler mice, a strain with hearing loss,vestibular defects, and pigmentation abnormalities (11). Mu-colipins constitute a family of cation channels with orthologs inhuman (MCOLN1), Drosophila melanogaster, and Caeno-

Table 5. Number of tags differentially represented in LK-3dand control libraries

cDNAs

MultipleMatches

NoReliableMatch

NoMatch Total

Knownfunction

Unknownfunction

LK-3d�C 76 (63%) 9 (8%) 10 (8%) 18 (15%) 7 (6%) 120C�LK-3d 18 (27%) 10 (15%) 1 (2%) 30 (45%) 7 (11%) 66Total 94 (51%) 19 (10%) 11 (6%) 48 (26%) 14 (7%) 186

Number of tags differentially represented in LK-3d and control libraries inthe different classes of identification through GenBank match. Tags matchingto a cDNA were subdivided into two classes on the basis of the presence orabsence of a functional characterization of the protein encoded by the cDNA.“Multiple Matches,” tags with �2 reliable identifications in GenBank; “NoReliable Match,” tag matching to cDNA(s) or EST(s) without poly-A signal orpoly-A tail; “No Match,” tag without any matching sequence in GenBank.





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Table 6. List of functionally identified tags present at higher level in LK-3d than in control library

Tag C LK LK/C Identification

AATAAATGTG 1 11 11.0 Plau: plasminogen activator (urokinase) (NM_008873)AAAGCACGCC 1 11 11.0 Gdf15: growth differentiation factor 15 (NM_011819)CCGTCCTCTA 1 10 10.0 Dctn2: dynactin 2 (NM_027151)TGAAAATTGG 1 9 9.0 Cox7c: cytochrome c oxidase, subunit VI1 (NM_007749)TCCTTCATCC 1 9 9.0 Rps27: ribosomal protein S27 (NM_027015)GCCTGCACAT 1 9 9.0 Mif: macrophage migration inhibitory factor (NM_010798)TATCCTAGCC 0 8 �8.0 Sui1-rs1: suppressor of initiator codon mutations, related sequence 1 (S. cerevisiae) (NM_011508) homologATGAACATCA 0 8 �8.0 Ndufb10: NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10 (XM_128594)ATCCAGTCCA 1 8 8.0 Jund1: Jun proto-oncogene related gene d1 (NM_010592)ATATTTTTTA 1 8 8.0 Pim3: proviral integration site 3 (NM_145478)TGGGCTGGTG 1 8 8.0 Muc1: mucin 1, transmembrane (NM_013605)AAGAATGGAC 1 8 8.0 Atp6v0d1: ATPase, H� transporting, V0 subunit D isoform 1 (NM_013477)TTGACCACTC 0 7 �7.0 Cfl1: cofilin 1, non-muscle (BC058726)TAAGTCGGCT 0 7 �7.0 Timp3: tissue inhibitor of metalloproteinase 3 (NM_011595)ACGCAACAGT 0 7 �7.0 Mcoln3: mucolipin 3 (AK035029)CGCTTGGCAG 0 7 �7.0 Psmb6: proteasome (prosome, macropain) subunit, beta type 6 (NM_008946)ACAGTATTGT 3 21 7.0 Tubb2: tubulin, beta 2 (NM_009450)AAAGGAGCTC 1 7 7.0 Fuca: fucosidase, alpha-L-1, tissue (BC003235)CTGTGTAGGA 1 7 7.0 Rab10: RAB10, member RAS oncogene family (BC052735)ATTTGACCCT 1 7 7.0 Scamp5: secretory carrier membrane protein 5 (NM_020270)TTGTGTGTTT 0 6 �6.0 Spt2: salivary protein 2 (NM8009268) or Pacsin2 protein kinase C and casein kinase substrate in neurons 2

(NM_011862)ATGCATTGTC 0 6 �6.0 Fos: FBJ osteosarcomma oncogene (NM_010234)AGGAGGGCCG 0 6 �6.0 Eps812: EPS8-like 2 (NM_133191)TTGCACCCCA 0 6 �6.0 Timm23: translocase of inner mitochondrial membrane 23 homolog (yeast) (NM_016897)TGCCCCCATA 0 6 �6.0 Atp6v1g1: ATPase, H� transporting, V1 subunit G isoform 1 (NM_024173)TGCAAACTTG 2 12 6.0 Ubiquinol cytochrome c reductase hinge protein (NM_025641)AAGACAAGTG 2 12 6.0 Got1: glutamate oxaloacetate transaminase 1, soluble (NM_010324)ACAACTGTAG 2 12 6.0 Ivns1abp: influenza virus NS1 binding protein (NM_054102)ATCAGTCTCT 2 11 5.5 Car15: carbonic anhydrase 15 (NM_030558)AGGCAGTTTC 0 5 �5.0 Tpd52: tumor protein D52 (NM_009412)TTAAATGGCG 0 5 �5.0 Cs: citrate synthase (NM_026444)GAACAGGAGC 0 5 �5.0 Adam9: disintegrin and metalloproteinase domain 9 (meltrin gamma) (NM_007404)TCAGCCAAGA 0 5 �5.0 Rasd1: RAS, dexamethasone-induced 1 (NM_009026)GGGGACAAAG 0 5 �5.0 Aes: amino-terminal enhancer of split (X73361)GCTGCCATCA 0 5 �5.0 Zpro: Z protein (NM_026617)AAGGCTCCCG 0 5 �5.0 Ndufs2: NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NM_153064)CAAATCAGGA 0 5 �5.0 Dnaja1: DnaJ (Hsp40) homolog, subfamily A, member 1 (BC057876)GTCAGTGACA 0 5 �5.0 Acad1: acetyl-coenzyme A dehydrogenase, long-chain (NM_007381)ACTCCAAGTT 0 5 �5.0 Mir16-pending: membrane interacting prot of RGS16 (NM_019580)CCTCCACCCT 0 5 �5.0 Rpl13a: ribosomal protein L13a (NM_009438) tag BAAGGAGCTCA 0 5 �5.0 Pold4: polymerase (DNA-directed), delta 4 (NM_027196)GGATGCAACG 0 5 �5.0 Ndufv1: NADH dehydrogenase (ubiquinone) flavoprotein 1 (NM_133666)TGATAAAATT 0 5 �5.0 Tcp1: t-complex protein 1 (NM_013686)GGACAGGCAG 0 5 �5.0 H13: histocompatibility 13 (BC034217)AACCTGGAGT 0 5 �5.0 Fth: ferritin heavy chain (NM_010239) tag BCAGCGCAGGG 3 15 5.0 Rpl4: ribosomal protein L4 (NM_024212)GGTGGGACCC 2 9 4.5 Ndufs5: NADH dehydrogenase (ubiquinone) Fe-S protein 5 (NM_134104)ATCGGCATGG 2 9 4.5 Atp5k: ATP synthase, H� transporting, mitochondrial F1F0 complex, subunit e (NM_007507)CGGGAGGTGT 4 17 4.3 Rpl36: ribosomal protein L36 (NM_018730)AAGGTGGGAG 4 16 4.0 Prdx5: peroxiredoxin 5 (NM_010021)AGGAGTTCAA 3 12 4.0 Slc22a4: solute carrier family 22 (organic cation transporter, OCTN1), member 4 (NM_019687)CCGCGAGGCC 2 8 4.0 Rps26: ribosomal protein S26 (NM_013765)CTAAACTCTG 2 8 4.0 Ncl: nucleolin (AF318184)ACCAGGAAAA 4 15 3.8 Atpi: ATPase inhibitor (NM_007512)AGCCAGGACC 3 11 3.7 Spnb3: spectrin beta 3 (XM_129139)CTGATGCCCA 5 18 3.6 Rpl32: ribosomal L32 protein (NM_172086)GGCAAGCTGG 5 17 3.4 Ndufa11-pending: NADH ubiquinone oxidoreductase subunit B14.7 (XM_128696)TCCTCAGCAT 3 10 3.3 Ccrn4l: CCR4 carbon catabolite repression 4-like (S. cerevisiae) (BC026940) or Mus musculus, clone

IMAGE:5368048, mRNA (BC030479)CTAGAGGAAA 4 12 3.0 Ndufs3: NADH dehydrogenase (ubiquinone) Fe-S protein 3 (BC027270)TTGTTGTGAG 11 29 2.6 Tde1: tumor differentially expressed 1 (BC011295)TGCAACTGTT 11 26 2.4 Rps28: ribosomal protein S28 (NM_016844)AGTGTTGAAA 12 28 2.3 Slc25a3: solute carrier family 25 (mitochondrial carrier; phosphate carrier), member-3 (NM_133668)TTGAGGTTAC 8 18 2.3 Rpl13a: ribosomal protein L13a (NM_009438) tag AGCCTGGAGAA 20 43 2.2 Rpl41: ribosomal protein L41 (NM_018860.2)AAGAAATACA 11 22 2.0 Slc25a5: solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5

(NM_007451)





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rhabditis elegans (CUP-5). Human MCOLN1 is a high-con-ductance calcium-sensitive calcium channel that is also perme-able to sodium and potassium (29). Mutations of MCOLN1 inhuman are responsible for mucolipidosis IV (3), a neurodegen-erative disease characterized by abnormal endocytosis (OMIM252650), whereas loss-of-function mutations of C. elegansCUP-5 leads to endocytosis defect and increased apoptosis (13,21). These defects are likely accounted for by the dysfunctionof intracellular mucolipin, and indeed, Mcoln3 is located in the

cytoplasm of inner ear hair cells (11). However, becauseMcoln3 is also located in the plasma membrane of stereocilia(11), it can be speculated that it might contribute to transmem-brane cation transport in kidney epithelial cells.

Renal acid/base balance in LK mice and expression ofrelated genes. Control of acid-base balance by the collectingduct is a function of �- and �-intercalated cells. The highoccurrence of the tag for �-intercalated cell-specific basolateralchloride/bicarbonate exchanger kAE1 and the absence of thatfor pendrin, the apical anion exchanger of �-intercalated cell(Table 4), is consistent with the fact that mouse OMCDs aredevoid of �-intercalated cells.

Present results confirm the previous observation (40) thatpotassium depletion induces no major change in acid-basestatus in mice, in contrast with the marked alkalosis thatprevails in potassium-depleted rats. In agreement with thisfinding, we found no change in expression of transcripts forcarbonic anhydrase II and kAE1, which constitute a transportmetabolon (51), the expression of which is controlled byacidosis and alkalosis (49, 55).

Nonetheless, results indicate changes in expression ofmRNAs encoding three proteins putatively involved in themaintenance of acid-base balance. The first two correspond toRhcg, the occurrence of which increased from 26 to 49 (P �0.008), and Rhbg, whose occurrence decreased, although notstatistically significantly, from 7 to 3 during potassium deple-tion (Table 4). Rhbg and Rhcg are non-erythroid members ofthe erythrocyte Rhesus protein recently cloned in the kidney(33, 34) that share homologies with Mep/Amt NH3/NH4

�

transporters in primitive organisms and plants (23). In ratOMCD, Rhbg and Rhcg are expressed at the basolateral andapical pole of intercalated cells (47), respectively, where theyare supposed to mediate NH3/NH4

� secretion. However, thereciprocal changes in expression of Rhcg and Rhbg transcriptsand the late time course of Rhcg induction (Fig. 3) suggest thattheir regulation in potassium depletion might not be related tothe regulation of transepithelial NH3/NH4

� secretion. The thirdtranscript corresponds to carbonic anhydrase 15, a putativenew member of carbonic anhydrases family for which the onlyavailable information concerns the cDNA sequence. In silicoanalysis of the cognate protein reveals the presence of a signalpeptide but no transmembrane or glycosyl-phosphatidyl-inosi-tol anchoring domain, suggesting that carbonic anhydrase 15might be secreted, as previously reported for carbonic anhy-drase 6 (41). If so, overexpression of carbonic anhydrase 15might not alter the function of OMCD cells but rather someextracellular process away from its synthesis site.

Table 6.—Continued

Tag C LK LK/C Identification

ACTCTATTTT 26 49 1.9 Rhcg: Rhesus blood group-associated C glycoprotein (NM_019799)AGTCTTTAAA 23 41 1.8 Rpl19: ribosomal protein L19 (NM_009078)TTGTGTCAGT 28 46 1.6 Atp1b1: ATPase, Na�/K� transporting, beta 1 polypeptide (X61433)

AAATAAAGTT 35 53 1.5Ldh2: lactate dehydrogenase 2, B chain (NM_008492) or 4921508E09Rik: RIKEN cDNA 4921508E09

gene (NM_025721)GTGGCAGTGG 328 468 1.4 Aqp2: aquaporin 2 (NM_009699)

For each tag, its sequence, its abundance in both control (C) and LK-3d (LK) library, the ratio value (LK/C), and its GenBank identification are given. Onlytags with �2 GenBank match to the nuclear genome are shown. Genes are identified according to the MGI nomenclature, including the symbol and full name.In some instances, a usual name was added in parenthesis. Tag A and tag B refer to tags corresponding to the last and penultimate 3�-most Sau3AI site. Thecomplete list of tags present at different levels in control and LK-3d libraries is presented in Supplemental Table S3.

Table 7. List of functionally identified tags present at lowerlevel in LK-3d than in the control library

Tag C LK C/LK Identification

ACAGTCCTTC 9 1 9.0 Ndufc1: NADH dehydrogenase(ubiquinone) 1, subcomplexunknown, 1 (NM_025523)

CCCTTCTACC 8 1 8.0 Anxa6: annexin A6 (NM_013472)CCAGGTGGAG 7 0 �7.0 Fkbp2: FK506 binding protein 2

(NM_008020)AAGAAGAACA 7 0 �7.0 Arf4: ADP-ribosylation factor 4

(NM_007479) or0610025G13Rik: RIKENcDNA 0610025G13 gene

GAGGAAACCA 7 1 7.0 Ndufa9: NADH dehydrogenase(ubiquinone) 1 alphasubcomplex, 9 (NM_025368)

AGGAGTACCA 7 1 7.0 Mbd2: methyl-CpG bindingdomain protein 2 (NM_010773)

CCACCCCAGT 5 0 �5.0 Ephx2: epoxide hydrolase 2cytoplasmic (NM_007940)

TGAAAGCCTG 5 0 �5.0 Ube2r2: ubiquitin-conjugatingenzyme E2R 2 (NM_026275)

ATCTAGAAAC 5 0 �5.0 Snx2: sorting nexin 2(NM_026386)

CAGGTCCTTC 5 0 �5.0 Pde4d: phosphodiesterase 4D,cAMP specific (NM_011056)

ATTTTGGTAT 5 0 �5.0 Lypla1: lysophospholipase 1(NM_008866)

CCAGGTAATT 5 0 �5.0 Arl6: ADP-ribosylation like6(NM_019665)

TGCGCTTCCA 19 7 2.7 H3f3a: H3 histone family, 3A(NM_008210)

AGCAAGCAGG 46 26 1.8 Actb: actin, beta, cytoplasmic(NM_007393)

ACTCTGGAGT 31 19 1.6 Igfbp7: insulin-like growth factorbinding protein 7 (NM_008048)

For each tag, its sequence, its abundance in both control (C) and LK-3d (LK)library, the ratio value (C/LK), and its GenBank identification are given. Onlytags with �2 GenBank match to the nuclear genome are shown. Genes areidentified according to the MGI nomenclature, including the symbol and fullname. The complete list of tags present at different levels in control and LK-3dlibraries is presented in Supplemental Table S3.





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Collecting duct hyperplasia and hypertrophy and expressionof related genes. Kidneys respond to numerous stimuli byincreasing the size of nephrons rather than their number. Suchincrease in size is mainly accounted for by cellular hypertro-phy, but cellular proliferation may also occur. In many circum-stances, renal cell hypertrophy is linked to increased ammoniaproduction (20, 28), which leads to an alkalinization of thelysosomal compartments and an inhibition of proteolysis andthereby to protein accumulation without progression of the cellcycle (15, 20). Hyperplasia is triggered by growth factors thatinduce the entry in the cell cycle and secondarily an increase inprotein synthesis and a reduction of proteolysis.

In the present study we observed that potassium depletioninduces both cellular hypertrophy (Fig. 1) and cellular prolif-eration (Fig. 2 and Table 3) in both principal and intercalatedcells of the OMCD. At the molecular level, the hypertrophy/hyperplasia process is attested by the “overexpression” oftranscripts encoding several ribosomal proteins, nucleolin,structural proteins (tubulin, the actin depolymerization factorcofilin, and spectrin �3), and proteins involved in theprocessing of the extracellular matrix [urokinase, tissueinhibitor of metalloprotease 3 (TIMP3)] and cell-matrixinteraction (ADAM9) (36). It is noteworthy, for example,that urokinase is involved in liver regeneration following

Fig. 3. Semi-quantitative RT-PCR analysis of mRNA ex-pression in OMCD. mRNAs from 1 mm of OMCD ofcontrol mice (control) and mice fed a potassium-depleteddiet for 3 days (LK-3d, hatched columns) 14 days (LK-14d,thin hatched columns) were reverse transcribed and ampli-fied by PCR using primers specific for growth differentia-tion factor 15 (Gdf15), urokinase, cofilin, organic cationtransporter 1 (OCTN1), and non-erythroid Rh protein(Rhcg) (see Supplemental Table S1). The DNA fragmentswere separated on 2% agarose gels and quantified using aPhosphorImager. Top: electrophoretical gels representativesof 3 animals from each group. Bottom: mean values fromseveral experiments. In each experiment, RNAs from atleast one control and one LK mouse were analyzed, andvalues were expressed as percent of the control value.Results are means � SE from several experiments indifferent animals (numbers of animals are in the columns).Statistical differences between control and LK-14d animalswere assessed by variance analysis: *P � 0.05, **P �0.005, ***P � 0.001.

Fig. 4. Expression profile of OCTN1 mRNAs along thenephron of control and LK-14d mice. mRNAs from 1 mm ofthe different segments of the nephron of control (solid blackcolumns) and mice fed a potassium-depleted diet for 14 days(gray columns) were reverse transcribed and amplified by PCRusing specific primers (see Supplemental Table S1). The DNAfragments were separated on 2% agarose gels and quantifiedusing a PhosphorImager. In each experiment, RNAs from atleast one control and one LK mouse were analyzed, and valueswere expressed as percent of the PCT value in control mouse.Results are means � SE from several experiments in differentanimals (numbers of animals are in the columns). Statisticaldifferences between control and LK-14d animals were as-sessed by variance analysis: *P � 0.05 and **P � 0.001. PCTand PST, proximal convoluted and straight tubule, respec-tively; MTAL and CTAL, medullary and cortical thick ascend-ing limbs of Henle’s loop, respectively; CCD, cortical collect-ing duct; NT, not tested.





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mass reduction. Also of interest, in relation with the remod-eling of cell membrane structure during potassium depletion(Fig. 1), is the “overexpression” of the transcript of Eps8-like 2 (42), a protein of the multimolecular complex thatlinks growth factor-induced activation of PI3-kinase withRac-induced reorganization of actin (25).

Potassium depletion is also associated with the “overexpres-sion” of transcripts previously found in various epithelialcancers, including kidney carcinomas: urokinase (44), TIMP3(44), the proto-oncogene kinase Pim3 (10), ferritin (8), tumorprotein D52 (6), mucin (19), cofilin (56), tumor differentiallyexpressed protein (45), macrophage migration inhibitory fac-tor, which promotes both tumor growth and tumor associatedangiogenesis (48, 53), and the two members of the AP-1transcription factor, JunD and Fos (57). Also, we observed“down-expression” of the tumor suppressor annexin 6 (18, 54)and of IGF-binding protein 7, also known as mac25, which isa growth-suppressing factor as well as an IGF-binding protein(43). With the exception of IGFBP-7, which may alter thedistribution along the nephron of IGF-I, a growth factor in-volved in potassium depletion-induced renal hypertrophy (14),all these genes may participate to the hypertrophy/hyperplasiaresponse of the OMCD to potassium depletion, but they likelydo not play a promoting role in these processes.

Present results also show that hyperplasia preceded hyper-trophy, as it was highest after 3 days of potassium depletion,when cell size was normal. This time course is consistent witha switch from hyperplasia toward hypertrophy through block-ade of the progression of the cell cycle before S phase. Twomechanisms have been reported to account for this hyperplasia/hypertrophy switch in renal cells. The first one is mediated bysignaling molecules, such as transforming growth factor-�(TGF-�) which stops growth factor-induced progression of cellcycle before entry into S phase. The second one is mediated byammonia, through inhibition of lysosomal function. BothTGF-� and ammonia action on switching EGF-induced hyper-plasia to hypertrophy require the activity of proteins of theretinoblastoma family (16, 17, 32). TGF-� inhibits growthfactor-induced phosphorylation of retinoblastoma family ofproteins via its action on cyclin E/cdk2 kinase (32), whereasthe mechanism linking lysosomal inhibition by ammonia withcell cycle remains unknown. Analysis of transcriptome pointsout several genes possibly involved in this hyperplasia/hyper-trophy switch in OMCDs. The first one is Gdf15, the expres-sion of which increased markedly between day 3 and day 14 ofpotassium depletion (Fig. 3), i.e., at the time of OMCD hyper-plasia. Thus, Gdf15, which is a member of TGF-� superfamily(4), may act as an autocrine growth factor in OMCD. Expres-sion of Gdf15 in hepatocytes is dramatically upregulated undercircumstances of liver injury and regeneration (22). Morerecently, Gdf15 was shown to prevent potassium depletion-induced apoptosis in cerebellar neurons (52), suggesting thatits expression might be controlled by potassium availability.Other genes possibly involved in the hyperplasia/hypertrophyswitch are those encoding Rhcg and Rhbg which likely alterammonia handling in OMCD intercalated cells (see above).Induction of the expression of OCTN1, which is also a trans-porter of weak organic bases and therefore may alter thelysosomal pH, may be related to the same process.

Comparison of human and murine OMCD transcriptomes.Because of the convenience of transgenesis in the mouse, thisspecies has become a must for physiological and pathophysi-ological studies. However, this raises the question of therelevance of the mouse as a model for human physiology.Comparison of mouse and human transcriptomes may providea partial answer to this question. From data in Table 4, whichcompares the abundance of some 40 transcripts in mouse andhuman OMCD, one can conclude that there is a good correla-tion between the abundance in the two libraries of a largemajority (70%) of tags, including cell-specific and -unspecifictags such as ENaC subunits and ribosomal proteins, respec-tively. However, the abundance of several markers of collect-ing duct cells is markedly different in mouse and humanlibraries. For example, expression of Rhcg and chloride/bicar-bonate exchanger kAE1 was �20-fold higher in mouse than inhuman OMCD. Also, there are marked discrepancies forAQP-2 and AQP-3: first, in human OMCD the abundance ofthe two water channels transcripts was similar, whereas inmouse OMCD abundance of AQP-2 transcripts was muchgreater than that of AQP-3. Second, expression of AQP-2 ismuch higher in mouse than in human OMCD. Whether thesedifferences reflect true species differences is not ascertained, asseveral epigenetic factors might account for them: 1) presentexperiments were performed on young adult mice, whereas thestudy in human was carried in patients aged 59 as a mean (7),and expression of AQP-2 decreases with aging (46); 2) humantissue was obtained from donors undergoing nephrectomy, andtherefore the surgical procedure may have altered gene expres-sion, particularly for AQP-2, the expression of which is highlydependent on the hydration status of the organism; and 3)although human mRNAs were extracted from healthy kidneyfragments, one cannot exclude that the neighboring tumor forwhich nephrectomy was performed and/or the presurgical che-motherapy given to the patients might have altered geneexpression.

Data in Table 4 also outline that some transcripts areexcluded from SAGE analysis. For example, calgizzarin,which is expressed at high level in mouse OMCD, cannot bedetected in human libraries because its cDNA lacks Sau3AIsite. The same holds for the channel-inducing factor CHIF, oneof the most abundant tags in the human OMCD library, whichcannot be detected in mouse library.

In summary, this study demonstrates the feasibility of dif-ferential analysis of transcriptome at the level of well-definednephron structures by using SADE. It also shows that, whencoupled to functional studies, transcriptome analysis may iden-tify candidate genes for distinct physiological processes. Fi-nally, this work provides a database accessible online for geneexpression in the mouse OMCD.

ACKNOWLEDGMENTS

We gratefully acknowledge the assistance of Emmanuelle Billon for se-quencing SAGE libraries and of Renee Gobin for electronic microscopy.

GRANTS

This work was supported by Centre National de la Recherche Scientifique(CNRS) and Commissariat a l’Energie Atomique grants to Unite de RechercheAssociee 1859 and CNRS grants to Unite Mixte de Recherche 7134.





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REFERENCES

1. Al-Awqati Q. Plasticity in epithelial polarity of renal intercalated cells:targeting of the H�-ATPase and band 3. Am J Physiol Cell Physiol 270:C1571–C1580, 1996.

2. Bakheet T, Williams BRG, and Khabar KSA. ARED 2.0: an update ofAU-rich element mRNA database. Nucleic Acids Res 31: 421–423, 2003.

3. Bargal R, Avidan N, Ben-Asher E, Olender Z, Zeigler M, Frumkin A,Raas-Rothschild A, Glusman G, Lancet D, and Bach G. Identificationof the gene causing mucolipidosis type IV. Nat Genet 26: 118–123, 2000.

4. Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, HeXY, Zhang HP, Donnellan M, Mahler S, Pryor K, Walsh BJ, Nichol-son RC, Fairlie WD, Por SB, Robbins JM, and Breit SN. MIC-1, anovel macrophage inhibitory cytokine, is a divergent member of TGF-betasuperfamily. Proc Natl Acad Sci USA 94: 11514–11519, 1997.

5. Buffin-Meyer B, Verbavatz JM, Cheval L, Marsy S, Younes-IbrahimM, Le Moal C, and Doucet A. Regulation of Na�,K�-ATPase in the ratouter medullary collecting ducts during potassium depletion. J Am SocNephrol 9: 538–550, 1998.

6. Byrne JA, Mattei MG, and Basset P. Definition of the tumor proteinD52 (TPD52) gene family through cloning of D52 homologues in human(hD53) and mouse (mD52). Genomics 35: 523–532, 1996.

7. Chabardes-Garonne D, Mejean A, Aude JC, Cheval L, Di Stefano A,Gaillard MC, Imbert-Teboul M, Wittner M, Balian C, Anthouard V,Robert C, Segurens B, Wincker P, Weissenbach J, Doucet A, andElalouf JM. A panoramic view of gene expression in the human kidney.Proc Natl Acad Sci USA 100: 13710–13715, 2003.

8. Cindolo L, Cantile M, Galasso R, Marsicano M, Napodano G, andAltieri V. Not traditional prognostic factors in human conventional renalcarcinoma. Minerva Urol Nefrol 53: 211–219, 2001.

9. Clarke L and Carbon J. A colony bank containing synthetic Col Elhybrid plasmids representative of the entire E. coli genome. Cell 9: 91–99,1976.

10. Deneen B, Welford SM, Ho T, Hernandez F, Kurland I, and DennyCT. PIM3 proto-oncogene kinase is a common transcriptional target ofdifferent EWS/ETS oncoproteins. Mol Cell Biol 23: 3897–3908, 2003.

11. Di Palma F, Belyantseva IA, Kim HJ, Vogt TF, Kachar B, andNoben-Trauth K. Mutations in Mcoln3 associated with deafness andpigmentation defects in varitint-waddler (Va) mice. Proc Natl Acad SciUSA 99: 14994–14999, 2002.

12. Elalouf JM, Buhler JM, Tessiot C, Bellanger AC, Dublineau I, and deRouffignac C. Predominant expression of �1-adrenergic receptor in thethick ascending limb of rat kidney. Absolute mRNA quantitation byreverse transcription and polymerase chain reaction. J Clin Invest 91:264–272, 1993.

13. Fares H and Greenwald I. Regulation of endocytosis by CUP-5, theCaenorhabditis elegans mucolipin-1 homolog. Nat Genet 28: 64–68,2001.

14. Flyvbjerg A, Marshall SM, Frystyk J, Rasch R, Bornfeldt KE, Arn-qvist H, Jensen PK, Pallesen G, and Orskov H. Insulin-like growthfactor I in initial renal hypertrophy in potassium-depleted rats. Am JPhysiol Renal Fluid Electrolyte Physiol 262: F1023–F1031, 1992.

15. Franch HA and Preisig PA. NH4Cl induced hypertrophy is mediated byweak base effects and is independent of cell cycle processes. Am J PhysiolCell Physiol 270: C932–C938, 1996.

16. Franch HA, Shay JW, Alpern RJ, and Preisig PA. Involvement of pRBfamily in TGF�-dependent epithelial cell hypertrophy. J Cell Biol 129:245–254, 1995.

17. Franch HA. Modification of the epidermal growth factor response byammonia in renal cell hypertrophy. J Am Soc Nephrol 11: 1631–1638,2000.

18. Francia G, Mitchell SD, Moss SE, Hanby AM, Marshall JF, and HartIR. Identification by differential display of annexin-VI, a gene differen-tially expressed during melanoma progression. Cancer Res 56: 3855–3858, 1996.

19. Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N,Burchell J, Pemberton L, Lalani EN, and Wilson D. Molecular cloningand expression of human tumor-associated polymorphic epithelial mucin.J Biol Chem 265: 15286–15293, 1990.

20. Golchini K, Norman J, Bohman R, and Kurtz I. Induction of hyper-trophy in cultured proximal tubule cells by extracellular NH4Cl. J ClinInvest 84: 1767–1779, 1989.

21. Hersh BM, Hartwieg E, and Horvitz HR. The Caenorhabditis elegansmucolipin-like gene cup-5 is essential for viability and regulates lyso-

somes in multiple cell types. Proc Natl Acad Sci USA 99: 4355–4360,2002.

22. Hsiao EC, Koniaris LG, Zimmers-Koniaris T, Sebald SM, Huynh TV,and Lee SJ. Characterization of growth-differentiation factor 15, a trans-forming growth factor beta superfamily member induced following liverinjury. Mol Cell Biol 20: 3742–3751, 2000.

23. Huang CH and Liu PZ. New insights into the Rh superfamily of genesand proteins in erythroid cells and nonerythroid tissues. Blood Cells MolDis 27: 90–101, 2001.

24. Imbert-Teboul M, Doucet A, Marsy S, and Siaume-Perez S. Alter-ations of enzymatic activities along the rat collecting tubule in potassiumdepletion. Am J Physiol Renal Fluid Electrolyte Physiol 253: F408–F417,1987.

25. Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Brachmann SM,DiFiore PP, and Scita G. Phosphatidylinositide 3-kinase activates Rac byentering in a complex with Eps8, Abi1, and Sos-1. J Cell Biol 160: 17–23,2003.

26. Kaissling B and Stanton BA. Adaptation of distal tubule and collectingduct to increased sodium delivery. I. Ultrastructure. Am J Physiol RenalFluid Electrolyte Physiol 255: F1256–F1268, 1988.

27. Kaufman AM and Kahn T. Potassium-depletion alkalosis in the rat.Am J Physiol Renal Fluid Electrolyte Physiol 255: F763–F770, 1988.

28. Kurtz I. Role of ammonia in the induction of renal hypertrophy. Am JKidney Dis 17: 650–653, 1991.

29. LaPlante JM, Falardeau J, Sun M, Kanazirska M, Brown EM,Slaugenhaupt SA, and Vassilev PM. Identification and characterizationof the single channel function of human mucolipin-1 implicated in mu-colipidosis type IV, a disorder affecting the lysosomal pathway. FEBS Lett532: 183–187, 2002.

30. Laroche-Joubert N and Doucet A. Collecting duct adaptation to potas-sium depletion. Seminars Nephrol 19 : 390–398, 1999.

31. Linas SL, Peterson LN, Anderson RJ, Aisenbrey GA, Simon FR, andBerl T. Mechanism of renal potassium conservation in the rat. Kidney Int15: 601–611, 1979.

32. Liu B and Preisig PA. TGF-�1-mediated hypertrophy involves inhibitingpRB phosphorylation by blocking activation of cyclin E kinase. Am JPhysiol Renal Physiol 277: F186–F194, 1999.

33. Liu Z, Chen Y, Mo R, Hui C, Cheng JF, Mohandas N, and Huang CH.Characterization of human RhCG and mouse Rhcg as novel nonerythroidRh glycoprotein homologues predominantly expressed in kidney andtestis. J Biol Chem 275: 25641–25651, 2000.

34. Liu Z, Peng J, Mo R, Hui C, and Huang CH. Rh type B glycoproteinis a new member of the Rh superfamily and a putative ammonia trans-porter in mammals. J Biol Chem 276: 1424–1433, 2001.

35. Loffing J, Le Hir M, and Kaissling B. Modulation of salt transport rateaffects DNA synthesis in vivo in rat renal tubules. Kidney Int 47:1615–1623, 1995.

36. Mahimkar R, Baricos WH, Visaya O, Pollock AS, and Lovett DH.Identification, cellular distribution and potential function of metallopro-tease-disintegrin MDC9 in the kidney. J Am Soc Nephrol 11: 595–603,2000.

37. Marples D, Frokiaer J, Dorup J, Knepper MA, and Nielsen S.Hypokalemia-induced downregulation of aquaporin-2 water channel ex-pression in rat kidney medulla and cortex. J Clin Invest 97: 1960–1968,1996.

38. Marsy S, Elalouf JM, and Doucet A. Quantitative RT-PCR analysis ofmRNAs encoding a colonic putative H,K-ATPase � subunit along the ratnephron: effect of K� depletion. Pflugers Arch 432: 494–500, 1996.

39. Marver D and Schwartz MJ. Identification of mineralocorticoid targetsites in the isolated rabbit cortical nephron. Proc Natl Acad Sci USA 77:3672–3676, 1980.

40. Meneton P, Schultheis PJ, Greeb J, Nieman ML, Liu LH, Clarke LL,Duffy JJ, Doetschman T, Lorenz JN, and Shull GE. Increased sensi-tivity to K� deprivation in colonic H,K-ATPase-deficient mice. J ClinInvest 101: 536–542, 1998.

41. Murakami H and Sly WS. Purification and characterization of humansalivary carbonic anhydrase. J Biol Chem 262: 1382–1388, 1987.

42. Offenhauser N, Borgonovo A, Disanza A, Romano P, Ponzanelli I,Iannolo G, DiFiore PP, and Scita G. The eps8 family of proteins linksgrowth factor stimulation to actin reorganization generating redundancy inthe Ras/Rac pathway. Mol Biol Cell 15: 91–98, 2004.

43. Oh Y, Nagalla SR, Yamanaka Y, Kim HS, Wilson E, and RosenfeldRG. Synthesis and characterization of insulin-like growth factor-binding





ownloaded from


protein (IGFBP)-7. Recombinant human mac25 protein specifically bindsIGF-I and -II. J Biol Chem 271: 30322–30325, 1996.

44. Patel SK, Ma N, Monks TJ, and Lau SS. Changes in gene expressionduring chemical-induced nephrocarcinogenicity in the Eker rat. Mol Car-cinog 38: 141–154, 2003.

45. Player A, Gillespie J, Fujii T, Fukuoka J, Dracheva T, Meerzaman D,Hong KM, Curran J, Attoh G, Travis W, and Jen J. Identification ofTDE2 gene and its expression in non-small cell lung cancer. Int J Cancer107: 238–243, 2003.

46. Preisser L, Teillet L, Aliotti S, Gobin R, Berthonaud V, Chevalier J,Corman B, and Verbavatz JM. Downregulation of aquaporin-2 and -3 inaging kidney is independent of V2 vasopressin receptor. Am J PhysiolRenal Physiol 279: F144–F152, 2000.

47. Quentin F, Eladari D, Cheval L, Lopez C, Goossens D, Colin Y,Cartron JP, Paillard M, and Chambrey R. RhBG and RhCG, theputative ammonia transporters, are expressed in the same cells in the distalnephron. J Am Soc Nephrol 14: 545–554, 2003.

48. Ren Y, Tsui HT, Poon RT, Ng IO, Li Z, Chen Y, Jiang G, Lau C, YuWC, Bacher M, and Fan ST. Macrophage migration inhibitory factor:roles in regulating tumor cell migration and expression of angiogenicfactors in hepatocellular carcinoma. Int J Cancer 107: 22–29, 2003.

49. Sabolic I, Brown D, Gluck SL, and Alper SL. Regulation of AE1 anionexchanger and H�-ATPase in rat cortex by acute metabolic acidosis andalkalosis. Kidney Int 51: 125–137, 1997.

50. Stanton BA and Kaissling B. Regulation of renal ion transport and cellgrowth by sodium. Am J Physiol Renal Fluid Electrolyte Physiol 257:F1–F10, 1989.

51. Sterling D, Reithmeier RA, and Casey JR. A transport metabolon.Functional interaction of carbonic anhydrase II and chloride/bicarbonateexchangers. J Biol Chem 276: 47886–47894, 2001.

52. Subramamiam S, Strelau J, and Unsicker K. Growth differentiationfactor-15 prevents low potassium-induced cell death of cerebellar granuleneurons by differential regulation of Akt and ERK pathways. J Biol Chem278: 8904–8912, 2003.

53. Sun B, Nishihira J, Suzuki M, Fukushima N, Ishibashi T, Kondo M,Sato Y, and Todo S. Induction of macrophage migration inhibitory factor

by lysophosphatidic acid: relevance to tumor growth and angiogenesis. IntJ Mol Med 12: 633–641, 2003.

54. Theobald J, Hanby A, Patel K, and Moss SE. Annexin VI has tumor-suppressor activity in human A431 squamous epithelial carcinoma cells.Br J Cancer 71: 786–788, 1995.

55. Tsurukoa S, Kittelberger AM, and Schwartz GJ. Carbonic anhydrase IIand IV mRNA in rabbit nephron segments: stimulation during metabolicacidosis. Am J Physiol Renal Physiol 274: F259–F267, 1998.

56. Unwin RD, Craven RA, Harnden P, Hanrahan S, Totty N, KnowlesM, Eardley I, Selby PJ, and Banks RE. Proteomic changes in renalcancer and co-ordinate demonstration of both the glycolytic and mito-chondrial aspects of the Warburg effect. Proteomics 3: 1620–1632, 2003.

57. Urakami S, Tsuchiya H, Orimoto K, Kobayashi T, Igawa M, and HinoO. Overexpression of members of the AP-1 transcriptional factor familyfrom an early stage of renal carcinogenesis and inhibition of cell growthby AP-1 gene antisense oligonucleotides in the Tsc2 gene mutant (Eker)rat model. Biochem Biophys Res Commun 24: 24–30, 1997.

58. Velculescu VE, Zhang L, Vogelstein B, and Kinzler K. Serial analysisof gene expression. Science 270: 484–487, 1995.

59. Virlon B, Cheval L, Buhler JM, Billon E, Doucet A, and Elalouf JM.Serial microanalysis of renal transcriptomes. Proc Natl Acad Sci USA 96:15286–15291, 1999.

60. Wittner M, Jounier S, Deschenes G, de Rouffignac C, and Di StefanoA. Cellular adaptation of the mouse cortical thick ascending limb ofHenle’s loop (CTAL) to dietary magnesium restriction: enhanced trans-epithelial Mg2� and Ca2� transport. Pflugers Arch 439: 765–771, 2000.

61. Wu X, George RL, Huang W, Wang H, Conway SJ, Leibach FH, andGanapathy V. Structural and functional characteristics and tissue distri-bution pattern of rat OCTN1, an organic cation transporter, cloned fromplacenta. Biochim Biophys Acta 1466: 315–327, 2000.

62. Zhang W, Kuncewicz T, Higham SC, and Kone BC. Structure, pro-moter analysis, and chromosomal localization of the murine H�/K�-ATPase �2 subunit gene. J Am Soc Nephrol 12: 2554–2564, 2001.

63. Zhang L, Zhou W, Velculescu VE, Kern SE, Hruban RH, HamiltonSR, Vogelstein B, and Kinzler KW. Gene expression profiles in normaland cancer cells. Science 276: 1268–1272, 1997.





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Plasticity of mouse renal collecting duct in response to potassium depletion

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