Relation between BK-a/b4-mediated potassium secretion and ENaC-mediated sodium reabsorption

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1 & 214 Interntionl Society of Nephrology sic reserch Reltion etween BK-/4-medited potssium secretion nd ENC-medited sodium resorption Donghi Wen 1, Ryn J. Cornelius 1, Dinelys Rivero-Hernndez 1, Yng Yun 1, Huqing Li 1, Aln M. Weinstein 2 nd Steven C. Snsom 1 1 Deprtment of Cellulr nd Integrtive Physiology, University of Nersk Medicl Center, Omh, Nersk, USA nd 2 Deprtment of Physiology nd Biophysics, Weill Cornell Medicl College, New York, New York, USA The lrge-conductnce, clcium-ctivted BK-/4 potssium chnnel, loclized to the interclted cells of the distl nephron, medites potssium secretion during highpotssium, lkline diets. Here we determine whether BK-/ 4-medited potssium trnsport is dependent on epithelil sodium chnnel (ENC)-medited sodium resorption. We mximized sodium potssium exchnge in the distl nephron y feeding mice low-sodium, high-potssium diet. Wild-type nd BK-4 knockout mice were mintined on low-sodium, high-potssium, lkline diet or low-sodium, high-potssium, cidic diet for 7 1 dys. Wild-type mice mintined potssium homeostsis on the lkline, ut not cid, diet. BK-4 knockout mice could not mintin potssium homeostsis on either diet. During the lst 12 h of diet, wild-type mice on either regulr, lkline, or n cid diet, or knockout mice on n lkline diet, were dministered miloride (n ENC inhiitor). enhnced sodium excretion in ll wild-type nd knockout groups to similr vlues; however, miloride diminished potssium excretion y 59% in wild-type ut only y 33% in knockout mice on n lkline diet. Similrly, miloride decresed the trns-tuulr potssium grdient y 68% in wild-type ut only y 42% in knockout mice on n lkline diet. tretment eqully enhnced sodium excretion nd diminished potssium secretion in knockout mice on n lkline diet nd wild-type mice on n cid diet. Thus, the enhnced effect of miloride on potssium secretion in wild-type compred to knockout mice on the lkline diet clrify BK-/4-medited potssium secretory pthwy in interclted cells driven y ENC-medited sodium resorption linked to icronte secretion. Kidney Interntionl (214) 86, ; doi:1.138/ki ; pulished online 26 Ferury 214 KEYWORDS: BK-/4; ENC; K secretion; mth modeling; N resorption; Correspondence: Steven C. Snsom, Deprtment of Cellulr nd Integrtive Physiology, Nersk Medicl Center, Omh, Nersk , USA. E-mil: ssnsom@unmc.edu Received 2 Octoer 213; revised 18 Novemer 213; ccepted 19 Decemer 213; pulished online 26 Ferury 214 The previling mechnism for K secretion in the distl nephron involves ction exchnge process with epithelil N chnnels (ENC) on the picl memrne of principl cells mediting N uptke in series with the solterl N K-ATPse, which ctively extrudes N nd trnsports K into the cell. Potssium secretion cn e enhnced y ldosterone, which increses the solterl N K-ATPse, 1 picl ENC, 2,3 nd picl renl outer medullry K chnnel (ROMK) 4 in principl cells. By this mechnism, the mximum K secreted to N resored rtio is.67, which is the rtio dictted y the two K per three N exchnge of the N K- ATPse. However, the Ynommi of South Americ ingest diet of very low-n nd high-k content, indicting mmmlin mechnism tht enhnces the K secreted per N resored rtio to vlue greter thn.67. It should e noted tht the urine of the Ynommi is very lkline, s shown y their very low urinry [Cl]. 5 We hypothesized tht the lklinity of their diet ws criticl for their ility to eliminte K with very low-n intke. 6 The mechnism for high rtio of K secretion to N resorption my involve secreting K independently of ENC-medited N resorption. 7 Supporting this notion, n isolted perfused corticl collecting duct (CCD) study showed tht lrge-conductnce, C-ctivted K chnnels (BK) medited luminl K exit. The N K-ATPse delivered the K into the cell ut N entered vi the solterl N H exchnger 8 insted of ENC. This mechnism for N-independent K secretion would require enhnced N K-ATPse in the solterl memrne. However, the mjority of BK chnnels reside in cid/se trnsporting interclted cells, which contin miniml N K-ATPse. 9,1 In interclted cells, the pore-forming BK- is ssocited with the BK-4 suunit. 11 Unlike wild-type, BK-4 knockout mice (KO) do not mintin K homeostsis when consuming high-k lkline diet. 6 The high-k diet cuses n increse in totl cellulr expression of BK-, vi ldosterone. 12 However, the BK- only ppers in the picl memrne, where it cn medite K secretion, in the presence of the BK-4 suunit. The BK-4 expression is enhnced when the urine is lkline. If the Ynommi hve dpted to low-n intke nd re secreting K independently of N resorption, then we would predict tht mice on low-n, high-k lkline diet would Kidney Interntionl (214) 86,

2 sic reserch D Wen et l.: ENC-dependent BK-/4-medited K secretion Tle 1 Comprison of vrious mesurements etween wild-type (WT) nd BK-4 knockouts (KO) on diets contining regulr chow (control), low N, high K, lkline (Alk) nd low N, high K, cidic (Acid) KW N V N Food N Wter N U[K] N U[N] N P[K] N P[N] N Uosm N Posm N Hct N UpH N Units mgm ml/dy gm/d ml/dy mmol/l mmol/l mmol/l mmol/l mosm/l mosm/l % WT control ±s.e.m WT-Alk ±s.e.m WT-Acid ±s.e.m KO-control ±s.e.m KO-Alk ±s.e.m KO-Acid ±s.e.m Arevitions: Hct, hemtocrit; KW, kidney weight; Posm, plsm osmollilty; s.e.m., stndrd error of men; U[K], urine [K]; U[N], urine [N]; Uosm, urine osmollity; UpH, urine ph; V, urine volume. mintin K lnce fter treting with miloride, which completely locks ENC-medited N resorption nd K secretion in isolted CCDs from rits on regulr diet. 13 However, we found tht mice use the BK-/4 of interclted cells to medite K secretion y mechnism tht is mostly dependent on ENC-medited N resorption in principl cells. We propose new model for K secretion tht involves recently discovered interclted cell trnsporters nd N recycling mechnism involving ctive pumping y principl cells nd pssive lek y interclted cells. RESULTS For ll experiments of this study, the tles show the electrolyte, osmollity, urinry volume, nd hemtocrit vlues red directly from the nlyticl instruments. The figures show comprisons of results clculted from these vlues. Role of lklinity in BK-/4-medited K secretion with low-n, high-k diet We previously showed tht WT mice on n lkline high-k diet mintined K lnce, wheres WT mice on n cidic high-k diet exhiited incresed plsm [K] nd diminished urinry K output. 6 The following experiments determined the importnce of lkline urine for hndling low-n, high- K diet. In these experiments, we compred K hndling etween WT nd KO on low-n, high-k, lkline diet (WT-Alk nd KO-Alk, respectively) nd etween WT nd KO on low-n, high-k, cid diet (WT-Acid nd KO-Acid, respectively) for 7 1 dys. As shown in Tle 1, the urine ph ws similrly lkline in WT-Alk nd KO-Alk, nd ws similrly cidic for WT-Acid nd KO-Acid. The plsm [K] (mmol/l) ws significntly elevted in KO-Alk, compred with WT-Alk; however, the plsm [K] of WT-Acid ws elevted to vlue not significntly different from the vlue for KO-Acid. As shown in Figure 1, the K clernce (ml/dy per gm kidney weight) for KO-Alk ws significntly lower, with vlue of ±85.2 (n ¼ 9), compred with vlue of ±112.8 (n ¼ 13) for WT-Alk. The K clernce ws depressed significntly in WT-Acid to 986.1±18.8 ml/dy per gm KW Alk K clernce Trns-tuulr K grdient Alk WT KO (n ¼ 6), vlue not significntly different from KO-Acid (738.4±85.4; n ¼ 4). The trns-tuulr K grdient () ws determined s mesure of K secretion in the connecting tuule (CNT) nd initil CCD, efore wter extrction (see Supplementry Figure S2 online). As shown in Figure 1, the of KO-Alk ws 17.2±1.4 (n ¼ 5), vlue significntly less thn the vlue of 23.4±1.9 (n ¼ 8) for WT-Alk; however, for KO-Acid, the vlue of 1.4±.2 (n ¼ 4) ws not different from the vlue of 1.2±.7 (n ¼ 6) for WT-Acid. These results indicte tht K homeostsis of mice on low-n, high-k diet is dependent on BK-/4-medited K secretion in concert with urinry lkliniztion. WT KO Acid Acid Figure 1 Br plots illustrting () K clernce nd () trnstuulr K grdient () for wild type (WT) nd knockout (KO) on Alk nd Acid diets. Clernce nd were clculted from vlues of Tle 1. Po.1 vs. WT; Po.5 vs. Alk. 14 Kidney Interntionl (214) 86,

3 D Wen et l.: ENC-dependent BK-/4-medited K secretion sic reserch Thizide- nd miloride-sensitive N nd K trnsport in the ASDN Experiments were designed to determine the N dependency of BK-/4-medited K secretion in mice on the low-n, high-k, lkline diet. For WT-Alk nd KO-Alk, olus of vehicle, hydrochlorothizide (), or miloride ws given to determine the mount of N-Cl co-trnsporter (NCC) medited N resorption vs. ENC-dependent N resorption/k secretion in ech genotype. (5 mg/kg) ws given for 12 h to determine whether WT or KO ws preferentilly resoring N vi NCC, rther thn ENC, in the ldosterone-sensitive distl nephron (ASDN). It ws shown previously tht doses of thizides X2 mg/kg elicited mximl ntriuretic response in mice. 14 The high-performnce liquid chromtogrphy mesurements of miloride in the plsm nd the clculted vlues of the distl lumen re shown in Supplementry Figure S1 online. We found tht vehicle tretment cused discourgement of drinking high volume of wter during the lst 12 h, therey cusing reduction in urinry flow nd smll increse in plsm [K]. These results show the importnce of mintining high urinry flow to mintin K lnce. Nevertheless, the thizide nd miloride dt provide informtion regrding the reltive vlues of miloride-sensitive N resorption nd K secretion with respect to vehicle in these mice. As shown in Tle 2 nd Figure 2, tretment incresed N excretion only in WT nd KO on control diets. As indicted y the decrese in plsm [N] nd the increse in hemtocrit, miloride, ut not, cused N nd volume depletion nd n elevtion of plsm [K] in WT nd KO on either control or Alk diet. Figure 2 revels tht miloride tretment decresed urinry K excretion (U K V; mmoles/dy per gm kidney weight) in WT control from 126.5±17.1 in vehicle (n ¼ 4) to 87.3±145.8 (n ¼ 4), nd in WT-Alk from ±69.8 in vehicle (n ¼ 1) to 189.2±173.6 (n ¼ 8), nd in KO-Alk from 2137.±365.2 in vehicle (n ¼ 6) to 154.3±22.4 (n ¼ 5). As shown in Figure 2, tretment incresed urinry N excretion (U N V; mmoles/dy per gm kidney weight) in WT control from vlue of 297.4±66.8 in vehicle (n ¼ 4) to 836.5±142.2 (n ¼ 6) with nd to 25.5±157.8 (n ¼ 4) with miloride. In WT-Alk, N excretion incresed from vehicle vlue of 26.2±3.2 (n ¼ 1) to 128.±56.8 (n ¼ 8) with miloride nd in KO from 19.3±1.4 (n ¼ 7) in vehicle to ±13.1 (n ¼ 5) with miloride. As shown in Figure 3, miloride tretment significntly decresed in WT control from 8.4±.6 (vehicle; n ¼ 4) to 3.1±.6 (n ¼ 4), in WT-Alk from 17.±1.3 (vehicle; n ¼ 1) to 5.5±.4 (n ¼ 8), nd in KO-Alk from 9.7±.9 (n ¼ 5) to 6.1±.1 (n ¼ 5), vlue not significntly different from tht of miloride-treted WT-Alk. The trns-tuulr N grdient (TTNG) ws determined s N resorption in the CNT nd initil CCD nd shown in Figure 3. In WT control, the TTNG incresed from.54±.7 (n ¼ 4) in vehicle to.133±.22 (n ¼ 7) with, nd to.32±4 (n ¼ 4) with miloride tretment. The TTNG of WT-Alk incresed from.41±.3 (n ¼ 7) in vehicle to.286±.17 (n ¼ 5) with miloride. The TTNG of KO-Alk incresed from.39±.3 (n ¼ 7) in vehicle to.23±.4 (n ¼ 6) with miloride. The fct tht there ws n undetectle effect of on N nd wter lnce in WT-Alk nd KO-Alk is consistent with high percentge of N ypssing NCC nd resoring vi ENC in exchnge for secreted K. These results show tht despite incresed miloride-sensitive N resorption in KO, the miloride-sensitive K secretion is reduced. We gve miloride to determine whether the defect in K secretion in WT-Acid resulted from reduced ENC-medited N resorption or decresed K secretion coupled with N resorption. When WT-Acid mice were given miloride, the U K V (Figure 4) decresed from ±23.3 (vehicle; n ¼ 5) to 615.2±83.6 (n ¼ 3) nd the U N V incresed from 21.9±2.3 (n ¼ 5) in vehicle to 441.1±3.9 (n ¼ 3) with miloride. As shown in Figure 4, miloride decresed the from 6.8±.4 (n ¼ 5) to 4.3±.2 (n ¼ 3) nd incresed the TTNG from.1±.15 (n ¼ 5) to.33±.2 (n ¼ 3). These results show tht the miloridesensitive N resorption in WT-Acid is similr to WT-Alk; however, the miloride-sensitive K secretion is considerly ttenuted s ws the KO-Alk. DISCUSSION The KO mice exhiit defective K secretion when consuming high-k, lkline diet 6, indicting tht BK-/4 meditesk secretion in these conditions. We fed mice low-n, high-k diet to mximize N K exchnge nd determine the N dependency of K secretion y BK-/4 in the ldosteronesensitive distl nephron. ws used to determine whether decresed K secretion in KO resulted from enhnced NCCmedited N resorption nd less delivery of N to ENC. tretment enled the determintion of ENCdependent N resorption nd K secretion y WT nd KO. In the current experiments, corticl K hndling hs een inferred from the computed from the finl urine. The, used frequently in previous studies 6,15 17, ws used in this study s determintion of K secretion in the CNT nd initil CCD. The ccurcy of this pproch my e open to question, especilly in view of the likelihood of K resorption long the collecting ducts (CD). Trditionlly, the formul for is derived y ssuming tht the only CD flux is tht of wter, in which luminl solutes re neither resored nor secreted. As shown in Supplementry Figure S2 online, this prolem is reconsidered from the perspective of two-solute system (nominlly KCl nd ure), in which there my e fluxes of oth. The resulting formul for the isotonic (corticl) [K] is the stndrd, plus n upwrd perturtion when there is K resorption, nd downwrd perturtion when there is ure resorption. In the presence of oth K nd ure resorption, these terms cn cncel, preserving the ccurcy of the stndrd term. Kidney Interntionl (214) 86,

4 sic reserch D Wen et l.: ENC-dependent BK-/4-medited K secretion Tle 2 Comprison of vrious mesurements etween WT nd 4KO on control, Alk, or Acid diets nd treted with vehicle (veh), hydrochlorothizide (), or miloride (mil) KW N V N U[K] N U[N] N P[K] N P[N] N Uosm N Posm N Hct N UpH N Units mgm ml/dy mmol/l mmol/l mmol/l mmol/l mosm/l mosm/l % WT-veh ±s.e.m WT ±s.e.m WT-mil ±s.e.m WT-Alk-veh ±s.e.m WT-Alk ±s.e.m WT-Alk-mil ±s.e.m KO-Alk-veh ±s.e.m KO-Alk ±s.e.m KO-Alk-mil ±s.e.m Arevitions: Acid, cidic; Alk, lkline; Hct, hemtocrit; KO, Knockout; KW, kidney weight; Posm, plsm osmollilty; s.e.m., stndrd error of men; U[K], urine [K]; U[N], urine [N]; Uosm, urine osmollity; UpH, urine ph; V, urine volume; WT, wild type. μmoles/dy per gm KW U k V μmoles/dy per gm KW U N V TTNG Figure 2 Summry r plots illustrting the effects of hydrochlorothizide () nd miloride vs. vehicle on () K excretion nd () N excretion for wild type (WT) on control diet (WT control), WT-Alk, nd knockout (KO)-Alk. Po.5 vs. vehicle. Po.5 vs. WT-Alk.. Figure 3 Summry r plots illustrting the effects of nd miloride vs. vehicle on () trns-tuulr K grdient () nd () trns-tuulr N grdient (TTNG) for wild type (WT) control, WT-Alk, nd knockout (KO)-Alk. Po.5 vs. vehicle. Po.5 vs. WT-Alk. The model clcultions of Supplementry Figure S2 online, Supplementry Tle S1 online lso llow exmintion of the ccurcy of the. In the second to lst column of the tle, the corticl K concentrtion is estimted from the finl urine using the stndrd ; the right-most column is determined y going ck into the luminl concentrtion profiles from the model nd selecting the K concentrtion t the point tht the lumen is isotonic 142 Kidney Interntionl (214) 86,

5 D Wen et l.: ENC-dependent BK-/4-medited K secretion sic reserch μmoles/dy per gm KW U K V nd U N V U K V U N V nd TTNG TTNG.2.1 Figure 4 Summry r plots illustrting the effects of hydrochlorothizide () nd miloride on () rtes of K nd N excretion nd () trns-tuulr K grdient () (left y-xis) nd TTNG (right y-xis) for wild type (WT)-Acid. Po.5 vs. vehicle. (3 mmol/l). Overll, greement of the estimte with the model vlue is t the 1% level, nd supports the pproch of the current study. Effects of tretment cused n expected N diuresis in mice on regulr diet. However, did not cuse n N diuresis in either WT or KO on Alk or Acid. These results show tht ll of the N in mice on Alk or KO ws resored y ENC, nd not NCC in the distl nephron. The fct tht ws ineffective regrdless of cid/se shows tht the high plsm [K] or its meditor, ldosterone, nd not the nion in the distl lumen, ws responsile for the incresed ENC- vs. NCC-medited N resorption. This result is consistent with previous study showing diminished ntriuretic response to thizides fter high-k feeding. 18 A high-k diet induces the WNK4-SPAK pthwy 19 nd signls the dephosphoryltion of NCC resulting in shift of distl N resorption from NCC to ENC in order to mximize N K exchnge. 2 However, our mthemticl modeling (see Supplementry Figure S2 online) using previously determined N resorptive permeilities reveled tht the NCC-medited N resorption ws not necessrily turned off ut ws merely undetectle compred with the overwhelmingly enhnced ENC-medited N resorption. N dependency of BK-/4-medited K secretion Our results demonstrted tht neither WT nor 4KO cn dpt to the low-n, high-k diet with n ENC-independent, N resoring mechnism tht cn mintin K, N, nd fluid lnce. Our findings differ from two studies tht demonstrted N-independent K secretion. Using isolted perfused rit CCD, it ws shown tht BK ws involved in mechnism to secrete K independently from N resorption. 8 Another study used miloride to demonstrte Nindependent K secretion in rts. 7 However, for mice dpted to low-n, high-k diet for 7 1 dys in the present study, miloride tretment reduced K secretion, elevted plsm [K], decresed volume, nd reduced plsm [N]. The plsm miloride concentrtion ws t lest 1.4 mmol/l during the course of 12 h, nd estimted in the CNT lumen t 22 6 mmol/l (Supplementry Figure S1 online). With n inhiitory constnt (Ki) of 1 nmol/l, 21,22 miloride will hve inhiited ENC y more thn 99% for the 12-h durtion. will not hve sustntilly inhiited the proximl tuule N H exchnger, with Ki 41 mmol/l. 23 Moreover, inhiition of the N H exchnger would result in incresed urinry ph, which ws not oserved. It is unlikely tht we did not oserve N-independent K secretion ecuse the flow ws not gret enough in the miloride-treted mice. A high luminl volume mintins low luminl K concentrtion, therey preventing high grdient for K resorption. 24,25 Moreover, high flow ctivtes BK ut cnnot ccount for lumen-negtive potentil tht drives K secretion, independent of N resorption. Our results re consistent with previous studies reveling tight coupling of ENC-medited N resorption for K secretion despite high distl flow Mechnism of enhnced K secretion in WT-Alk tretment reveled tht the defect of KO-Alk ws the inility to couple K secretion with ENC-medited N resorption to the mgnitude of WT-Alk. The mount of ENC-sensitive K secretion nd N resorption rtio (Ks/Nr) cn e estimted y the chnges in nd TTNG fter miloride tretment, nd ssuming tht the distl flow does not chnge sustntilly. With this ssumption, the Ks/Nr in WT-Alk is greter thn 1.5 nd the Ks/Nr in KO-Alk is less thn.5. This vlue for KO-Alk is ner the Ks/Nr of.4 in rits on control diet nd the Ks/Nr of.57 when rits were treted with deoxycorticosterone cette (DOCA; synthetic minerlocorticoid) in isolted rit CCD. 29 In nother study of isolted CCDs from DOCA-treted rits, the Ks/Nr ws.76, which exceeded the pump rtio of The DOCA-treted condition is different from dpting mice to the Alk diet, ecuse the niml on Alk will enhnce K secretion with miniml N delivery while lklinizing the urine. The fct tht ldosterone promotes HCO3 secretion vi pendrin 3 is consistent with neutrl exchnge of nions nd electricl coupling of N sorption with K secretion. Moreover, enhnced pendrin-medited HCO3 secretion is Kidney Interntionl (214) 86,

6 sic reserch D Wen et l.: ENC-dependent BK-/4-medited K secretion ssocited with incresed ENC-medited N resorption, 31 which would result in KHCO3 secretion driven y N K- ATPse in the principl cells (PC) nd H-ATPse in the interclted cells (IC). However, if Ks/Nr is greter thn one, then the mechnism must involve N recycling. The mechnism for very high Ks/Nr in WT-Alk my involve two novel trnsporters in the IC. As depicted in the cell model of Figure 5, the secretory N K-2Cl co-trnsporter 1 (NKCC1) ws reveled in the solterl memrne 32 nd the N-dependent Cl-HCO3 exchnger (NDCBE) in the picl memrne 33,34 of IC. With luminl [N] of 1 mmol/l, cellulr [N] of t lest 15 mmol/l, miniml N K-ATPse in IC, nd drive for HCO3 secretion, the chemicl grdients fvor N secretion/recycling. The solterl NKCC1 would trnsport K into the cell from B5 mmol/l (plsm) to 12 mmol/l (intrcellulr) using the chemicl grdients 3N + CI ENC 2K + ROMK PC 2 HCO 3 IC 2 H + N + 3CI K + BK-α/β4 3N + ATP ATP 2K + N + K + 2 CI Figure 5 Illustrtion of interction etween defined trnsporters of the corticl collecting duct tht cn explin how the N K- ATPAse of the principl cells (PC) cn drive BK-/4-medited K secretion in the interclted cells (IC). Secretion of HCO3 in exchnge for Cl vi pendrin rises the luminl [HCO3], s the luminl Cl is forced through the IC insted of through the prcellulr pthwy, where it would short-circuit the Vte. The greter resistnce of the tight-junction pthwy ugments the electronegtive lumen potentil nd driving force for K secretion. The lrge plsm to lumen chemicl grdient for N nd the genertion of intrcellulr HCO3 crete driving force for BK-medited K secretion nd NHCO3 secretion with N recycling to generte higher rtio of miloridesensitive K secretion per N resorption. fvoring N nd Cl cell entry. The solterl H-ATPse, which increses with high-k, lkline diet, 6 supports the cell electronegtive grdient 35 for Cl recycling vi the solterl Cl chnnel. 36 In isolted corticl collecting ducts, there is considerle pssive lek of N from th to lumen. 28,29 Reducing luminl [N] elow 15 mmol/l results in reversl of N trnsport cross the renl distl tuule. 25 After miloride tretment, the N concentrtion rises to 36 mmol/l nd the N trnsports into the cell with HCO3 recycling vi pendrin nd NDCBE, s previously descried. 33,34 This could explin the filure to lklinize the urine when miloride is given to Alk mice (see Tle 2). One cvet to this model is the notion tht NKCC1 is in the solterl memrne of -IC nd -IC, where they were first descried. 37 Becuse the lkline diet cuses n increse in -IC, compred with -IC, s indicted y n increse in pendrin expression, 12 we speculte tht recycling of N would occur in the -IC. CONCLUSION We conclude tht the BK-/4, loclized in interclted cells of the connecting tuules nd corticl collecting duct, medites K secretion y n ENC-medited, N-dependent pthwy. The BK-/4 pthwy ccounts for very high rtio of K secreted per N resored when mice re on low-n, high-k, lkline diet, similr to the diets of the Ynommi of South Americ. MATERIALS AND METHODS Animl studies Wild-type (WT; C57Bl/6, Chrles River, Wilmington, MA) nd BK-4 knockout mice (generously provided y R. Brenner) were mintined in ccordnce with the Institutionl Animl Cre nd Use Committee of the University of Nersk Medicl Center. For ll experiments, 12- to 2-week-old mice hd full ccess to wter. Mice were fed either regulr mouse chow (control;.6% K,.32% N) or specil diet (Hrln Tekld, Mdison, WI) for 7 1 dys efore killing. Specil diets were s follows: low-n, high-k with lkline nions (TD.7278; Alk; 5.% K with 5% of equl cronte/citrte/cl nd.1% N), or low-n, high-k with Cl s the counter nion (TD.975; Acid; 5.% K, 5.% Cl,.1% N). Twelve hours efore killing, suset of mice were treted with n intrperitonel (IP) olus of vehicle, hydrochlorothizde (; 5 mg/kg), t concentrtion of 1 mg/ml, or miloride (5 mg/kg), t concentrtion of 1 mg/ml. nd miloride were dissolved in polyethylene glycol. Tle 3 Comprison of vrious mesurements of WT mice on Acid diet nd treted with vehicle,, or miloride KW V U[K] U[N] P[K] P[N] Uosm Posm Hct UpH Units mgm ml/dy mmol/l mmol/l mmol/l mmol/l mosm/l mosm/l % Acid-veh ±s.e.m. (n ¼ 5) Acid ±s.e.m. (n ¼ 3) Acid-mil ±s.e.m. (n ¼ 3) Arevitions: Acid, cidic; Alk, lkline; mil, miloride; Hct, hemtocrit;, hydrochlorothizide; KW, kidney weight; Posm, plsm osmollilty; s.e.m., stndrd error men; U[K], urine [K]; U[N], urine [N]; Uosm, urine osmollity; UpH, urine ph; V, urine volume; veh, vehicle; WT, wild type. 144 Kidney Interntionl (214) 86,

7 D Wen et l.: ENC-dependent BK-/4-medited K secretion sic reserch After tretment, urine ws collected in metolic cges (Nlgene, Bellmore, NY), s previously descried. 1 The urinry N nd K concentrtions were nlyzed y flme photometer (Jenwy Clinicl PFP7, Stffordeshire, UK), s previously descried, 38 nd for ph nd osmollity using Model 215 ph meter (Denver Instruments, Bohemi, NY) nd Model 325 osmometer (Advnced Instruments, Norwood, MA), respectively. At the time of killing, we extrcted lood from the crotid rtery, mesured hemtocrit, nd centrifuged for mesurement of plsm [K], [N], nd osmollity. The trns-tuulr N nd K grdients were clculted y using the following formuls: urine [N] X plsm [Osm]/ urine [Osm] X plsm [N], nd urine [K] X plsm [Osm] / urine [Osm] X plsm [K], respectively. DISCLOSURE All the uthors declred no competing interests. ACKNOWLEDGMENTS This project ws funded y Ntionl Institutes of Dietes nd Digestive nd Kidney Diseses Grnts R1 DK29857 (AMW) nd RO1 DK7114 nd RO1 DK92474 (SCS), nd fellowship (11PRE75318) from the Americn Hert Assocition MWA Affilite (RJC). We pprecite the helpful comments of Dr Gerhrd Gieisch. Supporting grnts: R1 DK29857, RO1 DK7114, RO1 DK92474, nd AHA 11PRE SUPPLEMENTARY MATERIAL Figure S1. HPLC mesurement of miloride concentrtion in urine nd plsm. Figure S2. Mthemticl justifiction for. Tle S1. Urine concentrtions nd flows with lterntive model prmeters nd experimentl mneuvers potssium concentrtions when tuule fluid is isotonic. Supplementry mteril is linked to the online version of the pper t REFERENCES 1. Grg LC, Knepper MA, Burg MB. Minerlocorticoid effects on N-K-ATPse in individul nephron segments. Am J Physiol 1981; 24: F536 F Msilmni S, Kim GH, Mitchell C et l. Aldosterone-medited regultion of ENC lph, et, nd gmm suunit proteins in rt kidney. 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