Renal Tubular and Vascular Urea Transporters: Influence of Antidiuretic Hormone on Messenger RNA Expression in Brattleboro Rats

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1 J Am Soc Nephol 9: Renal Tubula and Vascula Uea Tanspotes: Influence of Antidiuetic Homone on Messenge RNA Expession in Battleboo Rats DOMINIQUE PROMENEUR, USE BANKIR, MING-CHANG HU, and MARIE-MARCELLE TRINH-TRANG-TAN Institut National de la Sante et de la Recheche M#{233}dicale, Unite 90, H#{244}pital Necke, Pais, Fance. Abstact. In the kidney, facilitated uea tanspot in pecise vascula and tubula stuctues is mainly involved in wate consevation. Thee uea tanspotes have been cloned: UT2- expessed in teminal inne medullay collecting duct (IMCD), U12- expessed in thin descending limb, and Ull 1 in descending vasa ecta. The effect of aginine vasopessin (AVP) administation on mrna expession of these thee tanspotes was examined in Battleboo ats with diabetes insipidus. V2 effects wee disciminated fom combined Vl + V2 effects by compaing teatments with l-deamino-8- D-AVP (ddavp) (selective V2 agonism) and AVP (V 1 and V2 agonism). Acute and chonic teatments wee studied. Abundance of specific mrna was assessed by quantitative Nothen blot analysis of RNA extacted fom two egions of inne stipe of oute medulla and fom two egions of inne medulla (IM). The esults show that mrna of these uea tanspotes ae diffeently egulated by AVP. ( 1) Long-tem teatment with eithe AVP o ddavp does not alte UI2- mrna in tip IM (teminal IMCD) except fo a tansient initial decease. (2) Unlike AVP, ddavp induces the appeaance of significant expession of UI2- mrna in base IM (initial IMCD), indicating a majo V2 effect. (3) UT2- mrna in deep inne stipe of oute medulla and base IM (thin descending limb of and loops, espectively) is pogessively upegulated with duation of AVP o ddavp teatment. (4) The much highe changes in UT2- and UT2- induced by ddavp compaed with AVP suggest that they ae dependent mainly on V2 agonism, and likely attenuated by Vi agonism. (5) UT1 I mrna expession in tip IM is equally depessed by AVP and ddavp, indicating that this vascula tanspote is also influenced by AVP and/o uine-concentating activity, via an indiect mechanism that emains to be detemined. High pemeability to uea in seveal tubula and vascula stuctues of the kidney had been suspected fo a time fom cleaance studies ( 1 ), and was late confimed by in vito micopefusion of dissected stuctues (2-5). These apid, caie-mediated uea movements wee localized in the teminal inne medullay collecting duct (IMCD), in some pats of thin descending limb of Henle s loop (TDL), and in descending vasa ecta (DVR). They ae all involved in uea movements, which ae elated to uinay concentating mechanism and wate consevation (6,7). The coesponding tanspotes have now been cloned in diffeent species (human, at, abbit) (8-13). Two tanspotes, expessed exclusively in the kidney, ae encoded by a single gene, UT2, but divege by altenative splicing: That in teminal IMCD is encoded by a 4-kb tanscipt of UT2 gene (UT2-), and that in TDL esults fom a 3-kb tanscipt (UT2-) (14). UT2- is the so-called vasopessin-egulated uea tanspote. UT1 1, initially identi- Received Octobe 14, Accepted Febuay 10, Coespondence to D. Maie-Macelle Tinh-Tang-Tan, Depatement de Biologie Cellulaie et Moleculaie, CEA/Saclay. Sevice de Biologic Cellulaie Gif-su-Yvette, C#{233}dex, Fance /0908- I 359$03.00/0 Jounal of the Ameican Society of Nephology Copyight 1998 by the Ameican Society of Nephology fled as the eythocyte uea tanspote (8), is found in seveal ogans including the kidney (8,15), whee it is expessed in endothelial cells of DVR ( 1 6). In pevious studies, egulation of UT2- and UT2- expession has been examined in nomal ats duing wate diuesis o sustained wate depivation, afte seveal weeks on a low potein diet, o afte adenalectomy (10, 17, 1 8). UT1 1 expession was examined only in whole kidney duing vaiations in the hydation state (IS). The aim of the pesent study was to evaluate the influence of the V 1 and V2 effects of aginine vasopessin (AVP) on the level of mrna of UT2-, UT2-, and UTI I in the kidney. We infused AVP (V 1 + V2) o 1 -deamino-8-d-avp (ddavp) (V2 only) in homozygous Battleboo ats unable to synthesize AVP, and theefoe exhibiting cental diabetes insipidus (DI). In such a model, the effects esulting fom exogenous antidiuetic homone (ADH) administation ae not obscued by changes in endogenous homone levels likely to occu in nomal ats. The time couse of the changes induced by ADH was examined afte -tem (6 o 16 h) o - - In two ecently published aticles, changes in UT nomenclatue have been poposed. The and tanscipts of UT2 have been called UTI and UT2, espectively ( 12). and UT1 1 has been enamed UT3 ( I I ). Fo this aticle, we decided to use the most commonly known designations fo UT. since a consensus has not been eached egading the new nomenclatue.

2 1360 Jounal of the Ameican Society of Nephology J Am Soc Nephol 9: , 1998 tem (5 d) teatment. mrna abundance was detemined by quantitative Nothen blot analysis of total RNA extacted fom diffeent medullay zones. Results show that AVP o ddavp administeed at doses inducing equivalent uine osmolalities does not influence U12- mrna in tip inne medulla (IM) (likely teminal IMCD), but does induce the appeaance of UT2- and UT2- mrna expession in base IM (likely in the moe initial pat of IMCD and of loop TDL, espectively). ddavp induces changes of much highe magnitude than AVP, suggesting that these egulations likely esult fom V2 actions of the homone and might be educed by V 1 actions. Finally, this study shows that UTI 1 is also influenced by ADH, pobably indiectly, because UTI 1 mrna levels in tip IM ae geatly and equally depessed by AVP and ddavp. Mateials and Methods Physiologic Study Adult male Battleboo ats (weighing appoximately 330 g) with cental DI, bed in ou laboatoy, wee housed in individual metabolic cages (Techniplast, Vaese, Italy) fo 7 d befoe (adaptation), and duing the entie expeiment. All ats had fee access to standad at chow (MS. Extalabo, Fance) and tap wate. AVP o ddavp (a selective V2 agonist of AVP; Feing, Malm#{246}, Sweden) ( I 9) was administeed at doses intended to induce equivalent uine osmolality. Because ddavp has a much e biological half-life than AVP, it was given at a much lowe dose. AVP (Sigma, St. Louis. MO) was infused at 5 pg/d (expeiment 1 ), and ddavp at 0.25.tgId (expeiment 2), using osmotic minipumps (model 1, Alzet Cop., Palo Alto, CA) pimed in 0.9% NaCl at 37#{176}Cfo 3 h befoe intapeitoneal implantation unde light ethe anesthesia. In each expeiment. thee goups of thee ats wee studied afte diffeent teatment duations: 6 h, 16 h, o 5 d. One additional sham-opeated at was studied at each time point in each expeiment. The data of the thee sham-opeated ats of each expeiment wee aveaged to fom a contol goup. Flow ate, osmolality, and uea concentation wee detemined in uine collected duing the 6- o 16 h-peiod of the -tem teatments, and duing the last 24 h of the -tem teatment. At the end of the expeiments, ats wee anesthetized with pentobabital, and a blood sample was collected fo measuement of hematocit, plasma osmolality (feezing-point osmomete, Roebling, Belin, Gemany), and uea concentation (modified Bethelot s method, Uea kit, BioM#{233}ieux, Lyon, Fance). Quantijication of mnrna Expession Kidneys wee emoved, weighed, and sliced. Tissue fagments wee dissected, at 4#{176}Cunde steeomicoscopic obsevation, fom medullay subegions: (1) the uppe half and the lowe half of the inne stipe of the oute medulla (supeficial and deep IS, espectively), because UT2- has been localized in deep, not in supeficial, half of -loop TDL (14,20); (2) the uppe thid and the lowe two-thids of the inne medulla (base and tip IM, espectively), because facilitated uea tanspot has been demonstated in teminal, and not in initial inne medullay, collecting duct (IMCD) (efeence 3) and because in situ hybidization has localized UT2- in tip, not in base, IM (I 2). Moeove, UT2- mrna has been evealed by Nothen blot in base, not in tip, IM (21). Total RNA was extacted fom 10 to 50 mg oftissue dissected fom each medullay subzone by the acid-phenol extaction method using RNA NOW 228 (Biogentex, Seabook, TX). Quantification of the message fo UT2- and UT2- was pefomed using the same at clone of 77 1 bp, which eveals UT2- as a 4.0-kb tanscipt and UT2- as a 3.0-kb tanscipt. UTI I mrna was evealed by a clone of 386 bp ( 15). Pobes wee adiolabeled by the andom-piming technique (Megapime labeling kit, Amesham), using [a-32p] dctp, to a specific activity of appoximately 2 X 10#{176} cpm/g edna. RNA samples (7 ;.tg) wee denatued and sepaated by electophoesis on agaose gel (1.2%) containing 0.6 M fomaldehyde. Equal RNA loading was veified by visual inspection afte coloation with ethidium bomide. RNA wee tansfeed ovenight fom gel to nitocellulose membanes (Hybond C-Exta, Amesham), which wee then baked in a vacuum oven (2 h, 80#{176}C).Blots wee placed in a glass hybidization tube containing 10 ml of medium composed of 5X saline-sodium phosphate-ethylenediamineteta-acetic acid (EDTA) (SSPE) (see composition below), 50% fomamide, 0.5% sodium dodecyl sulfate (SDS), 1 mg/mi each bovine seum albumin, Ficoll, and polyvinylpyolidone. and 40 j.g/ml denatued salmon spem DNA. Pehybidization was pefomed at 42#{176}Cfo 4 h in a HY- BRAIDTM oven. Then, the adiolabeled pobe was added to pehybidization medium (appoximately 4 X 106 cpmlml), and membanes wee incubated ovenight at 42#{176}C. Blots wee washed twice at 25#{176}C in 2X SSPE, 0.1% SDS fo 15 mm and once at 32#{176}C in 1 X SSPE, 0. 1% SDS fo 30 mm. The final wash was caied out at 55#{176}C fo UT2- and UT2-, and 42#{176}Cfo UTI 1, in 0.1 X SSPE, 0.1% SDS, fo 30 mm. The composition of I X SSPE was: 150 mm NaCl, 10 mm NaH2PO4, and 1 mm EDTA, ph 7.4. Blots wee exposed fo 2 to 4 d to autoadiogaphic film (Hypefilm-MP, Amesham) with intensifying sceens at -80#{176}C.Expession of mrna was quantified by densitomety (Deskan II and ScanAnalysis, Biosoft, Fance) afte scanning of the autoadiogams (Scan Jet, Hewlet Packad, Fance). Results ae expessed as abitay units of density. Statistical Analyses Results ae expessed as means ± SEM. Diffeences wee analyzed by two-way ANOVA (homone and teatment duation) followed, wheneve appopiate, by post hoc Fishe test. Results Uine and Blood Paametes ANOVA indicated that teatment with AVP o ddavp induced a significant decease in uine flow ate and an incease in uine osmolality and uea concentation (Figue 1 ). It also indicated that AVP at 5 p.g/d o ddavp at 0.25 tg/d induced changes of simila magnitude, except duing the vey fist hous of homone teatment (6 h) when ddavp induced significantly highe uine osmolality and uea concentation than AVP (Figue 1). Plasma uea concentation (13uea) was significantly inceased by AVP o ddavp, and without diffeence between the two homonal teatments. uea inceased abuptly duing -tem teatment, and tended to decease but emained highe than contol values theeafte (-tem teatment) (Figue 1). Hematocit and plasma osmolality wee not affected by AVP o ddavp teatment. Expession of UT2-Shot and UT2-Long mrna In contol Battleboo DI ats, UT2- mrna expession was significant only in deep IS (vey weak o absent in supeficial IS and base and tip IM), wheeas UT2- mrna was

3 . J Am Soc Nephol 9: , 1998 Regulation of Renal Uea Tanspotes by ADH 1361 URINE FLOW RATE 1500 URINARY UREA CONCENTRATION II *** E I Contol 6 hous 16 hous 5days Contol 6 hous 16 hous 5 days 2500 URINE OSMOLALITY 18 PLASMA UREA CONCENTRATION *** i c E Contol 6 hous 16 hous 5 days Contol 6 hous 16 hous 5 days Figue 1. Evolution of uine and plasma paametes in Battleboo (DI) ats duing teatment with aginine vasopessin (AVP) (dak bas) o l-deamino-8-d-avp (ddavp) (light bas). Each ba is the mean ± SEM of thee ats. Uine flow ate was significantly deceased, and uine osmolality and uine and plasma uea concentations significantly inceased by both AVP and ddavp teatment (ANOVA). ***P < by post hoc Fishe test. uea of ats teated fo 6 h with AVP o ddavp was significantly highe than uet of the espective contol goups. pesent in IM, with a much highe expession in tip than in base IM (Figue 2). ANOVA evealed that both AVP and ddavp significantly affected UT2- and UT2- expession in all enal medulla subegions, except fo UT2- in tip IM (Table 1). It also indicated that, compaed with AVP, ddavp exeted a significantly highe effect on UT2- expession in deep IS and base IM, and on UT2- expession in base IM (Table 1). U12- expession was pogessively inceased in supeficial and deep IS and in base IM, a with AVP o ddavp teatment duation (Figues 2 and 4). In supeficial IS, whee it is only scacely expessed, UT2- mrna was equally inceased by AVP o ddavp. In deep IS, AVP induced a theefold and ddavp an I i-fold incease in mrna levels of UT2- afte S d of teatment. In base IM, these changes wee nine- and 60-fold, espectively (Figue 4). Moeove, -tem AVP o ddavp induced UT2- expession in tip IM, whee it is usually absent. Howeve, the latte effect was not significant (Table 1). Duing the fist 16 h of teatment, UT2- expession was tansiently deceased in IM (base and tip) by AVP. Administation of ddavp induced a simila tansient decease of UT2- in tip IM only, but induced a huge elevation of UT2- expession in base IM afte 5 d of teatment, an effect not found afte -tem AVP administation (Figues 2 and 4). Expession of UTJ 1 mnrna In contol Battleboo DI ats, the expession of UT 1 1 was low in supeficial IS, intemediate in deep IS, and highest in whole IM (base and tip) (Figues 3 and 4). ANOVA evealed that teatment with ADH significantly affected UT1 I mrna expession, without diffeence between AVP and ddavp except in base IM (Table 1). Teatment with each homone induced an ealy decease in UTI 1 expession in all fou subegions. Theeafte, mrna levels in whole IS and in base IM etuned to peteatment levels ( 16 h), and late on (5 d) eached values highe than those of contol ats (Figues 3 and 4). In tip IM, UT1 1 mrna was geatly depessed by both homones fom 6 h and emained low theeafte (Figues 3 and 4).

4 I 362 Jounal of the Ameican Society of Nephology J Am Soc Nephol 9: , 1998 Uosm 212±35 (mosm I kg H20) Contol 6 hous 1 6 hous 5 days 640± ± ±79 IAVPI 1SISuP.. IMI L Deep #{149}. $,..t, 1j*- Base LTIP i Uosm 286± 43 (mosm I kg H20) Is IdDAVP 1 Sup. L Deep IM LTIp Base 1222± ± ± 107 Figue 2. Nothen blot analysis of mrna fo UT2- and UT2-, in supeficial and deep inne stipe (IS) of oute medulla, and in base and tip of inne medulla (IM), in contol Battleboo DI ats and in DI ats teated with AVP (top panel) o ddavp (bottom panel) fo vaious duations. Each lane, loaded with 7 g of total RNA, coesponds to a single at. Blots wee exposed to autoadiogaphic film fo 2 d. Table 1. Two-way ANOVA of mrna expession data#{176} UT2-Shot UT2-Long U Tl 1 Location Homone Teatment Duation Homone Teatment Duation Homone Teatment Duation Supeficial IS NS P < NS P < 0.01 Deep IS P < 0.01 P < NS P < Base IM P < 0.02 P < NS P < P < 0.05 P < Tip IM NS NS P < 0.05 P < NS P < U IS, inne stipe of the oute medulla; IM, inne medulla. Discussion Thee diffeent uea tanspotes have been cloned. Two of them ae expessed in kidney exclusively: UT2- located in TDL and UI2- expessed in teminal IMCD ( 10,14,20). UT1 1 is expessed in diffeent tissues including kidney (endothelial cells of DVR), bain (glial cells), testis, and spleen (I 1, 15, 16). In the kidney, all thee tanspotes ae involved in the uine concentating pocess: UT2- allows uea eabsoption in the deepest pat of the papilla fo intestitial uea accumulation, and UT2- and UTI 1 contibute to uea

5 J Am Soc Nephol 9: , 1998 Regulation of Renal Uea Tanspotes by ADH 1363 Contol 6 hous 16 hous 5 days Uosm (mosm I kg H20) 212± ± ± ± 79 I- Sup. Is L Deep IAVPI IMI LTjp Base.1., Uosm (mosm I kg H20) Sup. 286± ± ± ± 107 Is L Deep IdDAVP I IM LTIp Base Figue 3. Nothen blot analysis of mrna fo UT1 I in supeficial and deep inne stipe (IS) of oute medulla, and in base and tip of inne medulla (IM), in contol Battleboo DI ats and in DI ats teated with AVP (top panel) o ddavp (bottom panel) fo vaious duations. Each lane, loaded with 7 p.g of total RNA, coesponds to a single at. Blots wee exposed to autoadiogaphic film fo 4 d. ecycling, which limits the dissipation of the coticomedullay uea gadient diving wate out of the medullay collecting duct (7). The influence of diffeent hydation states (glucose wate diuesis, wate depivation, wate intake modulation) in nomal ats has been examined peviously (lo,l2,i5). The esults suggested that endogenous vasopessin level modulates mrna expession of UT2- and UI2-. Fo UT! 1 expession, the ADH effect has been examined in only one study, on whole kidney ( 15). Because vasopessin is endowed of V 1 and V2 moieties, the pupose of the pesent study was to evaluate the effect and inteaction of these two agonisms. Theefoe, the effect of ddavp, a selective V2 agonist, was compaed with that of AVP. Homones wee administeed to Battleboo ats genetically deficient in AVP, and the time couse of mrna expession of the thee uea tanspotes was assessed simultaneously on the same ats. In the pesent study, ddavp was administeed at a lowe dose than AVP because of its much e half-life. Despite diffeent doses, both homones induced equal changes in uine and plasma paametes, except duing the vey fist hous of teatment, when ddavp induced a highe antidiuesis than AVP, indicating that this effect depends mainly on V2 agonism. The elevated 1uea induced by eithe ADH teatment likely esulted fom deceased efficiency of uea excetion elated to deceased uine flow ate. The abundance of the uea tanspote mrna was assessed by Nothen blot analysis of total RNA extacted fom diffeent subegions of enal medulla. The supeficial half of inne stipe of oute medulla usually does not expess uea tanspotes. The deep half of inne stipe contains UT2- mrna in the lowe pat of loop TDL (20). The uppe thid (base) of inne medulla contains UT2- mrna of loop TDL, and a minute amount of UT2- mrna of initial IMCD. The

6 I 364 Jounal of the Ameican Society of Nephology J Am Soc Nephol 9: , UT2 - UT2 - UT11 Sup #{149} Is ue z 0 N- 0. C ) - C * I 1 I I IM Base 2 U) C I I I : : 100: 1 I I I I Tip Co 6h 16h 5d d0 6h 16h 6h 16h 5d Figue 4. Densitometic quantification of UT2-, UT2-, and UT1 1 mrna expession in fou enal medullay subegions of DI ats, teated by AVP (#{149}) o ddavp (0). Each point epesents the mean ± SEM of thee ats. Density is expessed as abitay units pe 7 tg of total RNA. #P < 0.05 and *D < 0.()Ol by post hoc Fishe test afte ANOVA. Note the 5 times highe scale fo UT2- in deep IS and base IM, than in supeficial IS and tip IM. lowe two-thids (tip) of inne medulla contains only UT2- of teminal IMCD. The two UT2 tanscipts wee evealed by the same cdna pobe and wee disciminated by Nothen blot analysis accoding to mrna length. Ou esults show that AVP and ddavp affected UT2-, UT2-, and UTI 1 diffeently. Vasopessin-stimulated uea flux in teminal IMCD has been demonstated by Sands and colleagues (2). Ou data showing no effect of poed ADH (AVP o ddavp) administation on UT2- mrna levels in teminal IMCD indicate that the vasopessin-dependent uea tanspot in teminal IMCD is not egulated at the level of gene tansciption. These obsevations diffe somewhat fom those of Smith et a!., who epoted a decease in UT2- mrna in inne medulla of nomal ats submitted to poed wate depivation and thus likely exhibiting high AVP plasma levels (10). The food avoidance as a esult of thisting and thus the maked fall in uea synthesis and excetion, vesus nomal food intake in the pesent study, could explain this diffeence. In teminal IMCD, the ADHegulated uea pemeability is govened by a apid cellula pocess without an incease in gene tansciption, at vaiance with wate pemeability that is enhanced by apid insetion of aquapoin 2 (AQP2) in apical membane, followed by a tem incease in AQP2 gene tansciption (22). Recent esults indicate that egulation of the IMCD uea tanspote by AVP does not depend on tafficking to the plasma membane (23). Ou study evealed an intiguing undeexpession duing the fist hous of homone teatment (AVP o ddavp) simultaneously with a pogessive incease in uine osmolality. This tansient fall in UT2- expession in teminal IMCD does not esult fom a geneal pocess of mrna undeexpession occuing in collecting ducts duing the building up of osmotic gadient, because mrna expession of G3PDH, an enzyme of glycolysis, the main metabolic pathway of collecting ducts (24), was not concomitantly deceased (data not shown). Ou data also show that ddavp, but not AVP, geatly inceases UT2- in base inne medulla of Battleboo ats. As a whole, ou data suggest a dual effect of ADH mediated by V2 action in IMCD. In teminal IMCD, ADH tigges apid cellula events that incease uea pemeability, wheeas in initial IMCD, ADH induces UT2- mrna abundance and possibly potein synthesis. These -tem effects of ADH would

7 J Am Soc Nephol 9: Regulation of Renal Uea Tanspotes by ADH I 365 esult in the expession of UT2- on a geate length of IMCD. Howeve, it should be noted that an incease in mrna abundance does not necessaily esult in an incease in potein synthesis. Actually, it has been ecently epoted by immunoblotting of total inne medullay membane factions, that infusion of AVP in Battleboo ats fo 5 d did not affect the amount of the 97-kD potein, the IMCD uea tanspote (25). Pevious studies by Imai and colleagues have shown the pesence of facilitated uea tanspot in TDL of -loop nephons of nomal at (4). The uea enty in TDL lumen seves to pevent uea dissipation fom medullay intestitium in venous blood. It has been ecently shown by immunohistology that UT2- encodes a uea tanspote situated in the teminal, but not the initial, potions of -loop descending thin limbs of nomal at (20). The same spatial distibution is found hee, in Battleboo DI ats, as suggested by the pesence of UT2- mrna in deep, but not in supeficial, half of IS. The pesent study shows that both AVP and ddavp geatly enhance UT2- mrna expession a with inceased uine concentating activity. As a whole, ou esults ae in ageement with those of Smith et a!., who epoted an incease in UT2- mrna in whole kidney and inne medulla in wate-esticted ats. (10). In the pesent study, the upegulation in deep IS could occu in - and/o -loop TDL. In base IM containing TDL of only -looped nephons, the upegulating effect of ADH is of geate magnitude than that obseved in IS. In tip IM, whee it is not nomally expessed, UT2- appeas afte -tem ADH infusion. These data suggest that ADH pefeentially upegulates the expession of the uea tanspote of -loop TDL, by inceasing gene expession a the entie length of the segment. In addition, it is emakable that the effect of ddavp lagely exceeds that of AVP. This indicates that this effect is likely mediated by the V2 moiety, and may esult fom some unknown pocess elated to the blunting Vi action (via postaglandin fomation) on V2 ecepto-mediated effect in collecting duct (26). If the huge incease in mrna expession afte -tem ddavp infusion induces an additional amount of potein fo uea tanspot, this could esult in futile exaggeated uea ecycling because uine osmolality and uine ove plasma uea concentations ae not inceased futhe by ddavp compaed with AVP. Because vasopessin eceptos have not been epoted in TDL, these inceases in messenge of the uea tanspote of TDL pobably esult fom an indiect action of the homone. UT1 1 is likely esponsible fo the facilitated uea tanspot of the DVR, demonstated by Pallone (5). This tanspote helps etun to the inne medulla the uea diffusing out of adjacent fenestated ascending vasa ecta (in vascula bundles), thus minimizing the dissipation of the medullay osmotic gadient in venous blood. UT1 1 has been ecently localized in endothelial cells of DVR and is supposed to be a constitutive, nonegulated uea tanspote unlike UT2- (16). Ou data show that UTI 1 gene expession can be modulated by ADH and/o uine-concentating activity. ADH (AVP as ddavp) induces diffeent effects on UT1 i gene expession in the diffeent medulla subegions. In IS and base IM, the ealy effect of ADH is a fall in UTI I mrna abundance, wheeas tem ADH tends to estoe nomal mrna levels. The tansient change could esult fom unknown ealy petubations occuing duing the tansition fom diuesis to antidiuesis, situations neve examined until now. In tip IM, on the othe hand, ADH induces a apid and sustained fall in UTI 1 mrna levels. These changes pobably do not esult fom changes in capillay netwok density because this decline occus vey apidly (within 6 h). They ae not due to a diect V 1 vascula effect because ddavp also induces the same changes and because V 1 eceptos have been localized in smooth muscle cells and not in endothelial cells whee UT1 1 is localized. Rathe, they could be the consequence of the hypeosmotic envionment and/o the deceased papillay blood flow esulting fom the opeation of the concentating medulla. It has been shown that duing antidiuesis, the decease in inne medullay blood flow is accompanied by an incease in oute medullay blood flow (27,28). In conclusion, the pesent study shows that ADH influences the expession of UT2-, UT2-, and UT1 1 diffeently. The ADH-dependent egulation of uea tanspot is likely achieved at the level of mrna expession fo UT2- in TDL, and fo UT2- in initial, but not in teminal, IMCD. Long-tem ADH administation induces expession of UT2- and UT2- on a geate length of loop TDL, and of IMCD, espectively. Ou data demonstate that these effects ae selectively dependent on V2 effect of the homone. 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