The connecting tubule is the main site of the furosemide-induced urinary acidification by the vacuolar H þ -ATPase

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1 originl rtile & 2006 Interntionl Soiety of Nephrology see ommentry on pge 1674 The onneting tuule is the min site of the furosemie-inue urinry iifition y the vuolr H þ -ATPse J Kovikov 1, C Winter 1, D Loffing-Cueni 2, J Loffing 2,3, KE Finerg 4, RP Lifton 4, E Hummler 2, B Rossier 2 n CA Wgner 1 1 Institute of Physiology n Center for Integrtive Humn Physiology, University of Zurih, Zurih, Switzerln; 2 Deprtment of Phrmology n Toxiology, University of Lusnne, Lusnne, Switzerln; 3 Deprtment of Meiine Antomy, University of Friourg, Friourg, Switzerln n 4 Deprtment of Genetis, Yle Meil Shool, New Hven, Connetiut, USA Finl urinry iifition is hieve y eletrogeni vuolr H þ -ATPses expresse in i-seretory interlte ells (ICs) in the onneting tuule (CNT) n the ortil (CCD) n initil meullry olleting ut (MCD), respetively. Eletrogeni N þ resorption vi epithelil N þ hnnels (ENCs) in the pil memrne of the segment-speifi CNT n olleting ut ells my promote H þ -ATPses-meite proton seretion y reting more lumen-negtive voltge. The ext loliztion where this suppose funtionl intertion tkes ple is unknown. We use severl mouse moels performing renl lerne experiments n ssesse the furosemie-inue urinry iifition. Inresing N þ elivery to the CNT n CCD y loking N þ resorption in the thik sening lim with furosemie enhne urinry iifition n net i exretion. This effet of furosemie ws olishe with milorie or enzmil loking ENC tion. In mie efiient for the IC-speifi B1 suunit of the vuolr H þ -ATPse, furosemie le to only smll urinry iifition. In ontrst, in mie with kiney-speifi intivtion of the lph suunit of ENC in the CCD n MCD, ut not in the CNT, furosemie lone n in omintion with hyrohlorothizie inue norml urinry iifition. These results suggest tht the B1 vuolr H þ -ATPse suunit is neessry for the furosemie-inue ute urinry iifition. Loss of ENC hnnels in the CCD n MCD oes not ffet this iifition. Thus, funtionl expression of ENC hnnels in the CNT is suffiient for furosemie-stimulte urinry iifition n ientifies the CNT s mjor segment in eletrogeni urinry iifition. Kiney Interntionl (2006) 70, oi: /sj.ki ; pulishe online 20 Septemer 2006 KEYWORDS: H þ -ATPse; epithelil N þ -hnnel; onneting tuule; furosemie; istl renl tuulr iosis (RTA); mouse Corresponene: CA Wgner, Institute of Physiology n Center for Integrtive Humn Physiology, University of Zurih, Winterthurerstrsse 190, Zurih CH-8057, Switzerln. E-mil: Wgner@ess.unizh.h Reeive 2 Jnury 2006; revise 28 June 2006; epte 18 July 2006; pulishe online 20 Septemer 2006 The kineys ply entrl role in mintining i se homeostsis y resoring ironte n exreting i equivlents generte y metolism. Finl urinry iifition tkes ple in the onneting tuule (CNT) n long the ifferent segments of the olleting ut (CD) nmely the ortil (CCD), outer meullry, n initil inner meullry olleting ut (MCD). These prts of the nephron re ompose of segment-speifi ells resoring N þ n sereting K þ n of interlte ells (ICs) involve in i se trnsport. Mny stuies hve esrie funtionlly n morphologilly t lest two types of ICs: type A n type B. 1 3 Type A ICs serete protons vi n pilly expresse vuolr H þ -ATPse. 4 This proton seretion is funtionlly ouple to the solterl nion exhnger AE1 relesing ironte into loo. Type B ICs reverse this proess, therey sereting ironte into urine n soring protons. 1,5 The role of thir sutype, non-a/non-b ICs, is not fully lrifie yet. Proton seretion through vuolr H þ -ATPses in the CNT n CCD is eletrogeni n is thought to e iniretly ouple to N þ resorption. 4,6 N þ resorption through the milorie-sensitive epithelil N þ hnnel (ENC) expresse in neighoring segment-speifi ells retes more lumen-negtive potentil whih hs een hypothetize to enhne H þ seretion y H þ -ATPses In the MCD, ENC expression is muh lower thn in the CNT n CCD, 11 the lumen potentil is more positive n H þ seretion is inepenent from N þ sorption. 12 The ENC onsists of three suunits terme,, g. 13,14 Loss-of-funtion muttions in either the humn,, org suunits of ENC use pseuohypolosteronism type 1 hrterize y severe neontl slt wsting, hyperklemi, n metoli iosis. 15 The suunit plys n essentil role in the trffiking of the hnnel to the ell surfe s well s forming prt of the pore. 13 Aprt from the kiney, ENC is lso expresse in istl olon n in upper n lower irwys where it lso meites N þ resorption. 14 Mie with omplete intivtion of ENC evelop ute postntl respirtory istress n ie within 40 h of irth from filure 1706 Kiney Interntionl (2006) 70,

2 J Kovikov et l.: semie-inue urinry iifition o r i g i n l r t i l e to ler their lungs from liqui. 16 Reently, novel mouse moel hs een generte with speifi intivtion of ENC in the entire CD ut not in CNT, 17 proviing tool for stuying soium lne s well s isturnes of proton seretion seonry to efetive N þ sorption. The vuolr H þ -ATPse is ompose of t lest 13 suunits of whih severl ell- n tissue-speifi isoforms exist. 4,18,19 The B suunit forms prt of the peripherl omin V 1 n two isoforms of this suunit, B1 n B2, hve een ientifie. Wheres the B2 isoform (ATP6V1B2) is lmost uiquitously expresse n ppers to serve in most ells house-keeping funtion, the B1 isoform (ATP6V1B1) hs more limite tissue istriution: speilize ells of the epiiymis, 20,21 the vs eferens, 21 the iliry oy of the eye, 22 the inner er, 23 n ll sutypes of ICs of the kiney. 20,24 Muttions in the gene enoing the B1 suunit result in istl renl tuulr iosis (RTA) in mn hrterize y the inility of the istl nephron to ppropritely iify the urine. 23 A mouse moel efiient for the Atp6v11 gene hs reently een generte with impire urinry iifition. 25 Vuolr H þ -ATPse tivity is lmost ompletely sent from the CCD. Interestingly, enhne luminl pperne of the B2 suunit hs een note in B1-efiient ICs, suggesting tht the B2 isoform oul ompenste for the loss of B1. 25 Nevertheless, Atp6v11-efiient mie evelop more severe metoli iosis with inppropritely lkline urine when hllenge with n orl i-lo (NH 4 Cl) hrteristi of RTA. Different sutypes of RTA hve een propose n lssifie se on linil tests. 26,27 These tests inlue orl NH 4 Cl- or prenterl N 2 SO 4 -loing n pplition of furosemie, whih ll le to n ute urinry iifition in helthy sujets ut not in ptients with speifi sutypes of RTA. semie inhiits the luminl N þ /K þ /2Cl otrnsporter in the thik sening lim, therey inresing the elivere frtion of N þ to the susequent nephron segments n stimulting N þ resorption through ENC. Ptients lking n pproprite urinry iifition fter furosemie pplition hve een lssifie s suffering from the voltge-efetive form of RTA. 9 In orer to test for n lolize the possile funtionl intertion etween N þ resorption n H þ seretion in the ifferent prts of the istl nephron, we performe lerne stuies in mie trete utely with furosemie, hyrohlorothizie, n milorie, n mesure urinry iifition n net i exretion (NAE). RESULTS Cololiztion of vuolr H þ -ATPse n ENC in neighoring ells in the olleting system Immunostining of mouse kiney with ntioies ginst linin D28k, B1 suunit of the vuolr H þ -ATPse, n the suunit of the ENC revele tht the B1 suunit of the H þ -ATPse is exlusively expresse in CNTs n CDs whih were ientifie on ount of their hrteristi loliztion in the ortil lyrinth n the meullry rys, respetively, n y their strong lelling with ntioies ginst the et suunit of the epithelil soium hnnel (ENC) n the lium-ining protein linin D28k (Figure 1). Higher mgnifition showe tht the B1 suunit is only expresse in sutype of epithelil ells lining CNT n CD tht o not express ENC or linind28k. This stining pttern is onsistent with n exlusive loliztion of the B1 suunit in ICs 28,29 whih re intermingle etween the ENC (n linin) positive CNT n CD (prinipl) ells. The effet of furosemie n milorie on urinry iifition In first group of ontrol nimls, the effet of ute furosemie n susequent milorie pplition on urinry Figure 1 Loliztion of vuolr H þ -ATPses n the ENC long the CNT n CCD in mouse kiney. ( ) Conseutive mouse kiney setions (/ n /) were stine ginst the B1 suunit of the vuolr H þ -ATPse, the suunit of the ENC, n the linind28k protein. Originl mgnifition 400. (e g) Higher mgnifition of CCD. ICs positive for the B1 suunit re mrke with, rrows inite segment-speifi ells stine for ENC n linind28k. Originl mgnifition 650. Kiney Interntionl (2006) 70,

3 o r i g i n l r t i l e J Kovikov et l.: semie-inue urinry iifition iifition ws teste. As shown in Figure 2, the initil urinry ph ws similr in ll groups efore the pplition of furosemie. A olus of furosemie (2 mg/g of oy weight (BW)) fter 60 min lowere the urinry ph with eing signifintly more ii fter 90 min (furosemie: ph versus ontrol: ph ), fter 120 min (furosemie: ph versus ontrol: ph ), n 150 min (furosemie: versus ontrol: ph ). The furosemie-inue urinry iifition ws ompletely olishe y the susequent ministrtion of milorie or enzmil, inhiitors of ENC tivity: ph 979 in the furosemie þ milorie group versus in furosemie-lone group fter 120 min s well s fter 150 min: ph for furosemie þ milorie versus ph for furosemie lone. The urine proution n exretion remine stle in the ontrol group uring the entire experiment, wheres it inrese in furosemie-trete nimls from ml/g fter 60 min to ml/g fter 90 min. A similr effet ws oserve in mie trete susequently with milorie, resulting in n inrese in urine output from 70.6 ml/g BW fter 60 min to ml/g BW fter 90 min. (Figure 2). The nlysis of urinry eletrolyte exretion revele tht the frtionl exretion (FE in %) of soium n hlorie inrese in mie trete with furosemie plus milorie (Figure 2 n e) with mximum fter 120 min (FE of soium % for furosemie þ milorie versus % in ontrol n FE of hlorie % in furosemie þ milorie versus % in ontrol). As summrize in Tle 1 n Figure 3, furosemie tretment resulte in slightly erese loo potssium onentrtion ( mmol/l in furosemie versus mmol/l in ontrol) n ugmente loo ironte onentrtion (270.7 with furosemie versus mmol/l uner ontrol). Thus, tretment of mie with furosemie le to the expete inrese in urinry output with n inrese in frtionl soium n potssium exretion ompnie y strong urinry iifition s esrie previously for rts n humns. 9 The stimultion of urinry iifition ws olishe y the ENC inhiitors milorie n enzmil Urine volume / oy weight (μl/g) FE (N) % 8.0 Control semie semie + milorie 0.5 Urine ph Amil Amil enzmil Control semie semie+milorie semie+enzmil FE (K) % FE (Cl) % Amil # # Amil Amil Figure 2 Effet of furosemie n milorie on urinry ph n eletrolyte exretion. () Applition of furosemie (2 mg/g BW) (n ¼ 17) use iifition of urinry ph ompre to untrete wil-type mie (Atp6v11 þ / þ, n ¼ 13). The effet of furosemie on urine ph ws reverse in mie trete susequently with milorie (5 mg/g BW) (n ¼ 14) or enzmil (2 mg/g BW) (n ¼ 6). () Urine proution n exretion remine stle in the ontrol group uring the experiment, wheres it inrese in furosemie- n milorie-trete nimls. () The FE (%) of potssium inrese in furosemie-trete nimls n ws reverse y the ition of milorie. (, e) The FE % of soium n hlorie showe n inrese of oth eletrolytes in the furosemie- n milorie-trete nimls. Po5 n Po01 versus ontrol, # Po5 etween furosemie tretment n furosemie þ enzmil/furosemie þ milorie. e 1708 Kiney Interntionl (2006) 70,

4 J Kovikov et l.: semie-inue urinry iifition o r i g i n l r t i l e ph (mmol/ l) Venous loo ph Soium/hlorie Control semie semie +milorie (mmol/ l) (mmol/ l) N + Cl Bironte Potssium Figure 3 Effet of furosemie n milorie on loo ph n eletrolytes. ( ) semie tretment resulte in erese loo potssium onentrtion s expete from the inrese urinry exretion. Bloo ironte levels showe teneny to e higher whih i not reh sttistil signifine. Po01 versus ontrol. onsistent with role of ENC in the furosemie-inue urinry iifition. A similr effet of milorie n the struturlly unrelte ENC loker trimterene on the furosemie-inue urinry iifition hs lso een reporte in rts. 30 The furosemie-inue urinry iifition is reue in mie efiient for the IC-speifi vuolr H þ -ATPse B1 suunit (ATP6V1B1) To emonstrte tht the furosemie-inue urinry iifition epene on the tivity of vuolr H þ -ATPses lolize in ICs, we use mouse moel efiient for the IC-speifi B1 suunit (Atp6v11 / ). Atp6v11 / furosemie-trete mie exhiite mrkely higher sl urine ph thn the Atp6v11 þ / þ furosemie-trete mie (ph versus ) n only mil urinry iifition upon furosemie ministrtion oul e oserve: ph versus fter 120 min (Figure 4). The resiul urinry iifition oserve in the B1-efiient mie my e owing to prtil ompenstion y the B2 isoform whih is more luminlly lolize in ICs of Atp6v11 / mie. 25 To ssess the effet of the tretments on NAE, we mesure totl NH 3 /NH þ 4 n totl phosphte exretion in urine n estimte NAE from these t s the urine smples were too smll to mesure titrtle iity. Both totl NH 3 /NH þ 4 n totl phosphte exretion inrese in response to furosemie s esrie previously in rts. 30 Applition of furosemie inrese NAE from mmol/l/mg/l retinine in ontrol nimls to mmol/l/mg/l retinine (Figure 4). In mie given milorie, NAE showe teneny to e lower (P ¼ 8). In ontrst, in enzmil-trete mie n in the B1-efiient mie, NAE ws signifintly lower (enzmil: mmol/ l/mg/l retinine, B1 KO: mmol/l/mg/l retinine). Atp6v11 / furosemie-trete mie lso h strikingly higher urine output thn the Atp6v11 þ / þ furosemie-trete mie fter 60 min efore the furosemie ministrtion: ml/g BW versus ml/g BW (Figure 4). semie ministrtion le to more profoun iuresis in Atp6v11 / mie: 8.97 ml/g BW versus ml/g BW fter 90 min (Figure 4). In ition, Atp6v11 / mie h signifintly lower sl FE of potssium thn Atp6v11 þ / þ mie ( versus %) (Figure 4), sl FE of soium (671 versus %) (Figure 4e), n sl FE of hlorie (471 versus %) (Figure 4f) pointing to efet in urine onentrtion. semie ministrtion further revele signifint ifferene in the FE of potssium, wheres the FE of soium n hlorie i not iffer etween Atp6v11 / n Atp6v11 þ / þ mie (Figure 4e n f). As summrize in Tle 1 n Figure 5, nlysis of loo eletrolytes revele ifferenes in potssium n ironte levels. The CD-speifi loss of ENC expression in Cre mie oes not ffet the furosemie-inue urinry iifition n iureti response Cre mie lk the lph suunit of the ENC in the CCD n MCD ut not in the onneting segment. 17 We use these mie to test for the furosemie-stimulte urinry iifition. Bsl urine output, urine ph, n eletrolyte ontent were not signifintly ifferent etween ontrol mie ( ) n mie with the speifi ltion of ENC expression (Cre ) (ompre Tles 1 n 2). semie ministrtion use urinry iifition in oth genotypes to similr extent: n Cre mie: ph versus fter 120 min (Figure 6). Also, estimte NAE t the time point 150 min ws similr in oth groups of mie (Figure 6; mmol/l/mg/l retinine versus Cre mmol/l/mg/l retinine, P ¼ 0.31). No ifferene etween n Cre mie in urine output oul e etete ( ml/g BW versus ml/g BW fter 90 min.) (Figure 6). semie lso file to unmsk possile ifferene etween Kiney Interntionl (2006) 70,

5 o r i g i n l r t i l e J Kovikov et l.: semie-inue urinry iifition Urine ph Urine volume/oy weight (μl/g) e FE (N) % Atp6v11+/+ ontrol Atp6v11+/+ furosemie Atp6v11 / furosemie NAE/re (mg/l) Atp6v11+/+ furosemie Atp6v11 / furosemie FE (K) % f FE (Cl) % Control semie semie + milorie semie + enzmil Atp6v11 / furosemie 30 min 150 min Figure 4 Effet of furosemie on urine ph n eletrolyte exretion in mie efiient for the B1 vuolr H þ -ATPse suunit (Atp6v11 / ). () Atp6v11 / reeive furosemie (2 mg/g BW) (n ¼ 9) n urine ph iifie only milly. Po01 versus ontrol. () Estimte NAE t the time point 30 min n 150 min. NAE inrese fter furosemie pplition n the rise in NAE ws signifintly reue fter pplition of enzmil n in the Atp6v11-efiient mie (Po5 signifint ifferene etween furosemie-trete nimls n other groups). () semie ministrtion le to more profoun iuresis in Atp6v11 / in omprison to Atp6v11 þ / þ mie. ( f) The FE of N þ n Cl ws similr in oth genotypes; however, the FE % for potssium ws lower in B1-efiient mie. Vlues of ontrol n furosemie-trete nimls from Figure 2 re shown gin for omprison. Po5 n Po01 versus Atp6v11 þ / þ trete with furosemie. ph N + Venous loo ph Soium/hlorie Cl Bironte Potssium Atp6v11+/+ontrol Atp6v11+/+ furosemie Atp6v11 / furosemie Figure 5 Effet of furosemie on loo ph n eletrolytes in Atp6v11 / mie. ( ) Bloo potssium levels were lso lower in B1-efiient mie fter tretment with furosemie. Bironte ws higher thn in untrete wil-type nimls. Vlues from wil-type nimls re shown gin in Figure 3 for omprison. Po5 n Po01 versus ontrol. n Cre mie in the FE of soium: % versus % fter 120 min (Figure 6e n Tle 2). However, loo eletrolyte nlysis revele signifintly lower loo ironte onentrtion ( mmol/l in versus mmol/l in Cre (Tle 2 n Figure 7). Thus, fter pplition of furosemie, no signifint ifferene ws foun in the iureti response, urinry iifition, n eletrolyte exretion suggesting 1710 Kiney Interntionl (2006) 70,

6 J Kovikov et l.: semie-inue urinry iifition o r i g i n l r t i l e Tle 1 Summry of loo ph, gs, n eletrolytes of wil-type mie left untrete (ontrol) n wil-type n Atp6V11-efiient mie trete with furosemie n milorie t the en of the experimentl perio Atp6v11 +/+ ontrol Atp6v11 +/+ furosemie Atp6v11 +/+ furosemie milorie Atp6v11 / furosemie ph pco 2 (mmhg) HCO K N Cl Serum retinine (mg/l) Weight (g) CrCl/BW 30 min (ml/min/g) CrCl/BW 90 min (ml/min/g) CrCl/BW 120 min (ml/min/g) NH 3 /NH + 4 (mm)/retinine (mg/l), 30 min NH 3 /NH + 4 (mm)/retinine (mg/l), 150 min NAE (mm/retinine (mg/l), 30 min NAE (mm/retinine (mg/l), 150 min BW, oy weight; CrCl, retinine lerne; NAE, net i exretion. Mrks signifint ifferenes Po5, Po01. Tle 2 Summry of loo ph, gs, n eletrolytes of n Cre mie efiient for the lph ENC suunit trete with furosemie n hyrohlorothizie furosemie Cre furosemie furosemie HCT Cre furosemie HCT ph PCO 2 (mmhg) HCO K N Cl Serum retinine (mg/l) Weight (g) CrCl/BW 30 min (ml/min/g) CrCl/BW 90 min (ml/min/g) CrCl/BW 120 min (ml/min/g) NH 3 /NH + 4 (mm)/retinine (mg/l), 30 min NH 3 /NH + 4 (mm)/retinine (mg/l), 150 min NAE (mm/retinine (mg/l), 30 min NAE (mm/retinine (mg/l), 150 min BW, oy weight; CrCl, retinine lerne; NAE, net i exretion. Mrks signifint ifferenes Po5, Po01. tht preserve expression of ENC in the CNT is suffiient to mintin the furosemie-inue urinry iifition. In orer to inhiit possile ompenstory inrese in N þ sorption in the istl tuule vi the thizie-sensitive N þ /Cl otrnsporter, furosemie ws pplie together with hyrohlorothizie. Figure 8 shows tht the urinry ph of Cre mie i not iffer signifintly from the urine ph of mie efore furosemie n hyrohlorothizie (furo þ HCT) ministrtion. After ministrtion of furosemie together with hyrohlorothizie (furo þ HCT), Cre mie erese their urine ph to the sme extent s the mie: ph versus ph fter 120 min. Urine output, frtionl eletrolyte exretion n systemi eletrolyte, n loo gs sttus were not istinguishle etween oth mouse lines (Figures 8 n 9 n Tle 2). DISCUSSION In the present stuy, we investigte the funtionl intertion etween renl N þ resorption through ENC n H þ seretion y vuolr H þ -ATPses in the onneting segment n CCD. Both ENC n vuolr H þ -ATPses ontining the B1 suunit isoform re expresse together in the sme nephron segments, nmely CNT n CCD in neighoring ells. H þ seretion y vuolr H þ -ATPses is eletrogeni n thought to e iniretly ouple to N þ resorption. 31 Aoring to this hypothesis, N þ resorption through ENC retes more lumen-negtive trnstuulr voltge whih in turn woul enhne H þ seretion y vuolr H þ - ATPses. Thus, the furosemie-inue inrese in N þ elivery to the CNT n CCD n the susequent resorption of this elivere frtion through ENC my result in urinry iifition meite y vuolr H þ -ATPses. Kiney Interntionl (2006) 70,

7 o r i g i n l r t i l e J Kovikov et l.: semie-inue urinry iifition ph Urine volume/oy weight (μl/g) e FE (N + ) % Cre NAE/re (mg/l) FE (K + ) % f FE (Cl ) % Cre 30 min 150 min Cre Figure 6 Effet of furosemie on urine ph n eletrolyte exretion in n Cre mie. () The effet of furosemie on urinry iifition in (n ¼ 10) n Cre (n ¼ 12) mie. semie ministrtion i not show ny ifferene in urine ph etween n Cre mie. () No ifferene in the furosemie-inue inrese in the estimte rte of NAE ws oserve etween n Cre trete with furosemie. () Urine volume/bw in (n ¼ 11) n Cre (n ¼ 10) mie. No ifferene etween n Cre mie in urine output ws reporte. ( f) FE of potssium, soium, n hlorie in (n ¼ 6) n Cre (n ¼ 7) mie. semie-inue inrese of FE of potssium, soium, n hlorie i not iffer etween n Cre mie. ph Venous loo ph N + Soium/hlorie Snn1 loxlloxcre Cl Bironte Potssium Figure 7 Effet of furosemie on loo ph n eletrolytes in n Cre mie. Cre mie h signifintly lower ironte onentrtion. Other prmeters were not ifferent. Po5 versus. Severl types of efets in istl renl tuulr iifition hve een lssifie se on provotive tests using infusions ontining sulfte, ironte, or phosphte, orl NH 4 Cl loing or furosemie pplition n the respetive response in urinry ph, net i, n eletrolyte exretion s well s the ility to sustin loo urine PCO 2 grient. 32 At lest three types of RTA hve een istinguishe, seretory type with efet suppose in the proton-seretory proton pumps, 1712 Kiney Interntionl (2006) 70,

8 J Kovikov et l.: semie-inue urinry iifition o r i g i n l r t i l e Urine volume/oy weight (μl/g) FE (N + ) % Urine ph HCT +HCT +HCT Cre FE (K + ) % e FE (Cl ) % Cre HCT +HCT Figure 8 () Effet of furosemie (2 lg/g BW) n hyrohlorothizie (HCT, 5 lg/g BW) on urinry iifition in (n ¼ 6) n Cre (n ¼ 6) mie. Urine ph of Cre mie i not iffer signifintly from the urine ph of mie neither efore nor fter furosemie þ HCT ministrtion. ( e) Urine output n FE of potssium, soium, n hlorie in furosemie- n hyrohlorothizie-trete (n ¼ 5) n Cre (n ¼ 7) mie. No ifferene etween n Cre mie in urine output, FE of potssium, soium, n hlorie ws reporte. ph N + Venous loo ph Soium n hlorie Cre Cl Bironte Potssium Figure 9 Bloo ph n eletrolytes in furosemie- n hyrohlorothizie-trete (n ¼ 5) n Cre (n ¼ 7) mie. No signifint ifferene etween n Cre mie in loo ph n eletrolytes oul e oserve. k-lek type where H þ or HCO 3 my lek k, n voltge-efetive type where the inility to proue n i urine my result from the filure to proue fvorle lumen-negtive potentil filitting proton seretion. 9,26,33 Consistent with these funtionl lssifitions, pplition of milorie to ptients n experimentl nimls reues urinry proton seretion. 9,32,34 LiCl hs een use s nother tool to inue the voltge-epenent efet; however, severl Kiney Interntionl (2006) 70,

9 o r i g i n l r t i l e J Kovikov et l.: semie-inue urinry iifition stuies hve inite tht the efets inue y LiCl n milorie re ifferent. 35 The efet inue y ministrtion of LiCl n e restore y N 2 SO 4 infusion 32 n is ssoite with ltere expression levels of H þ /K þ -ATPses n H þ - ATPses. 36 In our experiments, furosemie inue strong urinry iifition. This iifition pprently epens on the tivity of vuolr H þ -ATPses ontining the B1 isoform s no or only mil iifition oul e oserve in Atp6v11 efiient mie. Thus, vuolr H þ -ATPses re the min meitor of the furosemie-inue urinry iifition. The result lso suggests tht H þ /K þ -ATPses, the other potentil i-seretory pumps, o not ply n importnt role s n i-seretory mehnism uner these experimentl onitions. However, we oserve lower urinry potssium exretion in the B1-efiient mie. The mehnism is presently unknown. A stimultion of H þ /K þ - ATPses, potentilly inue y higher urinry flow rte, oul ontriute to this effet. The effet of furosemie ws lunte y milorie inhiiting ENC-epenent N þ sorption s evient from the inrese frtionl N þ exretion n erese frtionl K þ -exretion. In seprte group of nimls, the onsequene of the geneti ltion of ENC hnnel funtion ws teste in Cre mie. These nimls lk the lph suunit of ENC whih is essentil for ENC funtion n results in no etetle ENC hnnel funtion n loss of luminl loliztion of et n gmm suunits in the CCD n outer meullry CD. 17 In the CNT, however, ENC funtion n loliztion hs remine norml. 17 In the ontrol n ENC-efiient mie, furosemie inue norml urinry iifition with similr inrese in frtionl N þ, Cl, n K þ exretion. In orer to rule out ompenstory inrese in N þ sorption y the thizie-sensitive N þ /Cl otrnsporter, we lso teste for the omine effet of furosemie n hyrohlorothizie whih use stronger urinry iifition n iureti response to the sme extent in oth groups of nimls. Thus, the furosemie-inue urinry iifition involves the tivity of n milorie- n enzmil-sensitive mehnism, most likely ENC. In view of the norml pity of Cr mie to resor N þ n to respon to furosemie n hyrohlorothizie, it ppers tht the resiul ENC funtion in the CNT my e suffiient for the furosemie-inue urinry iifition. However, it nnot e ompletely rule out tht milorie ts lso on mehnism istint from ENC n tht this mehnism is still opertive in the Cre mie. Bse on mthemtil moelling of trnsport proesses involve in i seretion n eletrolyte trnsport long the CD, it hs een reently propose tht the genertion of more lumen-negtive potentil y the tion of ENC oul hrly ount for hnges in vuolr H þ -ATPse tivity. 7 Weinstein 7 rgue tht the hnge in trnstuulr voltge is minor ompre to the ility of the pump to e tive even ginst lrger voltge grient, n propose tht milorie interts lso with other mehnisms. These lultions were me for the CCD n my not fully pply for the CNT whih ws ientifie here s the min trget of furosemie. However, our results o not ompletely rule out tht some of the urinry iifition inue y furosemie took ple in the CCD n tht milorie te on mehnism istint from ENC. This mehnism, however, remins to e ientifie n lol opposing ph grients must lso e tken into onsiertion. In ition, Hropot et l. 30 reporte tht the struturlly unrelte trimterene hs similr inhiitory effet on the furosemie-inue urinry iifition strongly suggesting tht ENC is the ommon trget. This ientifies the CNT s the mjor segment meiting furosemie-inue urinry iifition. Similr onlusions hve een rehe with respet to the N þ -soring pity of the CNT versus CCD. 17 Further experiments estlishing omplete geneti ltion of ENC expression long the entire CNT n CD will e helpful to prove this hypothesis. In summry, the furosemie-inue urinry iifition requires vuolr H þ -ATPses ontining the B1 suunit, is inhiite y milorie, n is not ltere in mouse moel with preserve ENC funtion in the CNT ut loss of ENC funtion in the CCD n outer meullry olleting ut. The results point to hitherto unreognize mjor role of the CNT in eletrogeni H þ -seretion n the urinry iifition inue y furosemie. MATERIALS AND METHODS Animls Experiments were performe on Atp6v11 þ / þ n Atp6v11 / mie 25 ) n Cre n mie. 17 They were mintine on stnr iet n h free ess to rinking wter. All experiments were performe oring to Swiss Animl Welfre lws n pprove y the Lol Veterinry Authority. The genertion, reeing, n genotyping of Atp6v11 /,, n Cre hs een esrie previously. 17,25 Bloo n urinry prmeters Mie were nesthetize intrperitonelly (ketmine 1 ml/g BW n xylzine 1 ml/g BW), ple on hete tle mintine t 371C n theter ws ple into the urinry ler. Urine ws ollete every 30 min uring the whole perio of 3.5 h for the mesurement of urine volume, ph, soium, hlorie, potssium, n retinine onentrtions. To prevent ehyrtion, mie were onstntly supplemente with 125 mm NCl n 25 mm NHCO 3 (ph 7.4) oring to their urinry output. At the en of the experiment, heprinize mixe venous loo ws ollete for the mesurement of ph, loo gses, soium, potssium, hlorie, n retinine onentrtions. After n initil reovery phse n olletion of seline vlue t 30 min, t 60 min Atp6v11 þ / þ, Atp6v11 /,, n Cre mie were ministere furosemie 2 mg/g BW suutneously; some of the n Cre mie were lso given hyrohlorothizie 5 mg/g BW together with furosemie. Two sugroups of Atp6v11 þ / þ mie reeive itionlly milorie 5 mg/g BW suutneously or enzmil 2 mg/g BW suutneously fter 90 n 120 min of the experiment. All rugs were finlly issolve in 1714 Kiney Interntionl (2006) 70,

10 J Kovikov et l.: semie-inue urinry iifition o r i g i n l r t i l e 125 mm NCl þ 25 mm NHCO 3. Control mie reeive only 125 mm NCl þ 25 mm NHCO 3. Urinry ph ws immeitely mesure; urine n loo eletrolytes n other loo prmeters (pco 2, po 2 ) were etermine using loo gs nlyzer (ABL 505, Riometer, Copenhgen, Denmrk). Urinry retinine ws mesure y the Jffe metho. Serum retinine ws etermine y n enzymti retion (F DAOS metho, Wko Cretinine F L-Type kit, Wko Chemils GmH, Germny). Totl urinry NH 3 /NH 4 þ ws mesure using the Berthelot protool, 37 totl urinry phosphte using ommeril kit (Sigm, Buhs, Switzerln). NAE ws estimte from the onentrtion of totl NH 3 /NH 4 þ, totl phosphte s the mjor titrtle i, n urine ph using the Henerson Hssellh eqution n ssuming loo ph 7.4. Immunohistohemistry Fixtion of mouse kineys n tissue proessing for immunohistohemistry ws performe s esrie previously. 28 Conseutive ryosetions (4 5 mm thik) of the fixe kineys were ple on hrom-lum geltine-ote slies, preinute with 10% norml got serum in phosphte-uffere sline, n fterwrs inute overnight t 41C with mouse-nti-hiken linin D28k ntioies (Swnt Bellinzon, Switzerln, ilution 1/ in phosphte-uffere sline-1% ovine serum lumin) tht ws pplie together with either rit-nti-rt ENC ntiser 17 (ilution 1/1000 in phosphte-uffere sline-1% ovine serum lumin) or rit-nti-rit B1 suunit of H þ -ATPse 20 (ilution 1/1000 in phosphte-uffere sline-1% ovine serum lumin). Bining sites of the primry ntioies were revele with fluoresein isothioynte-ouple got-nti-mouse immunogloulin G n Cy3-ouple got-nti-rit immunogloulin G (oth from Jkson Immuno Reserh Lortories, West Grove, PA, USA). 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