Role of luminal anion and pH in distal tubule potassiumsecretion - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：J. B. O.Amorim, M. A.Bailey, R.Musa-Aziz, G.Giebisch,  G.Malnic作者单位：1 Basic Science Department, Faculdade de Odontologiade São José dos Campos, and Departmentof Physiology and Biophysics, Instituto de CiênciasBiomédicas, Universidade de São Paulo, 05508-900 São Paulo, Brazil; and Department of Cellularand Molecular Physiology
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      【摘要】
   Potassium secretory flux( J K ) by the distal nephron is regulated bysystemic and luminal factors. In the present investigation, J K was measured with a double-barreledK electrode during paired microperfusion of superficialsegments of the rat distal nephron. We used control solutions(100 mM NaCl, pH 7.0) and experimental solutions in whichCl had been replaced with a less permeant anion and/orpH had been increased to 8.0. J K increasedwhen Cl was replaced by either acetate (~37%), sulfate(~32%), or bicarbonate (~62%), and also when the pH of thecontrol perfusate was increased (~26%). The majority (80%) ofacetate-stimulated J K was Ba 2  sensitive, but furosemide (1 mM) further reduced secretion (~10% oftotal), suggesting that K -Cl cotransport wasoperative. Progressive reduction in luminal Cl concentration from 100 to 20 to 2 mM caused increments in J K that were abolished by inhibitors ofK -Cl cortransport, i.e., furosemide and[(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminalperfusion fluid also increased J K even in thepresence of Ba 2 , suggesting that this effect cannot beaccounted for only by K channel modulation ofK secretion in the distal nephron of the rat.Collectively, these data suggest a role forK -Cl cotransport in distal nephronK secretion.
      【关键词】 distal tubule collecting duct potassium secretion postassiumchloride cotransport
   INTRODUCTION" K& _& x8 {3 a9 O+ H/ M& f) \1 d
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A LARGE NUMBER OF FACTORS modulate potassium (K ) secretion by theinitial and cortical collecting tubules ( 10 ). Luminal modulators include changes in the delivery of Na ( 12, 26 ), the transepithelial potential difference( 9 ), the concentration of Ca 2  ( 21 ), the presence of impermeant anions, and theconcentration of Cl relative to that of other anions( 6 ).
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K secretion rises sharply when impermeant anions arepresent in the lumen of the distal tubule ( 27 ). Earlierstudies attributed this effect to the increase in the lumen-negativetransepithelial potential difference generated by persistentNa reabsorption in the face of the decrease in anionconductance resulting from the replacement of permeableCl by a much less permeant species, such as sulfate( 5, 11 ). In vivo voltage-clamp experiments confirm thetransepithelial potential difference as an important modulator ofK secretion in the distal tubule ( 9 ).Nevertheless, the stimulatory effect of sulfate on K secretion persists even when the lumen-negative potential difference iscollapsed by amiloride ( 27 ) and is not affected by theinclusion of Ba 2  in the perfusate ( 6 ).Therefore, it is not possible to attribute the effect of anions onK secretion solely to changes in transepithelial potentialdifference, and it has been proposed that substitution ofCl by sulfate may enhance electroneutral K secretion by creating a favorable transmembrane gradient for K -Cl cotransport from cell to lumen( 6 )." k( Z/ X! R8 ^* Q
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Similar conclusions can be reached from studies in humans. Infusions ofNa sulfate have a greater kaliuretic effect than do those of NaCl( 4 ), although this is only apparent when the urinaryCl concentration is to modulate K excretion is criticallydependent on urinary Cl concentration provides strong, ifcircumstantial, evidence for K -Cl cotransport as a pathway for K secretion in the distalnephron. These studies further reveal that infusions of Na bicarbonatestimulate K excretion to a greater extent than do those ofNa sulfate ( 4 ). In contrast to the actions of sulfate, thekaliuretic effect of bicarbonate was not dependent on a concomitantlylow urinary Cl concentration. These observations wouldsuggest that bicarbonate ions wield an additional influence onK secretion and thus excretion.& m" t4 S+ L# n+ P

We have used stationary microperfusion of superficial segments of therat distal tubule to investigate the role of luminal anions and pH,either separately or in combination, in modulating K secretion. We observed that K secretion was greater underconditions in which Cl was replaced by either sulfate oracetate. This increment in K secretion could be abolishedby known inhibitors of K -Cl cotransport,suggesting that the effect of both sulfate and acetate was indirect,attributable to low luminal Cl concentration. Replacementof Cl with bicarbonate induced a further increment inK excretion. Our experiments suggest that modulation ofK -Cl cotransport by luminal fluid pH plays akey role in this action of bicarbonate on K secretion.
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METHODS
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Male Wistar rats (wt range 180-320 g), kept on a standardrat chow (plasma K concentration = 4.0 ± 0.7 mM), were anesthetized with Inactin (100 mg/kg ip) and preparedsurgically for micropuncture experiments. During the experiments, bodytemperature was maintained at 37°C, and isotonic saline,containing 3% mannitol, was infused intravenously at a rate of 4 ml/h.

Stationary microperfusion. The technique employed in these experiments ( 1 ) isillustrated in Fig. 1. A proximal tubulewas impaled by a double-barreled micropipette, and FD&C green controlsolution was injected from barrel P to localize distaltubule segments with one or more surface loops (Fig. 1 A ).Once the K -selective microelectrode (see below) had beeninserted into the last surface loop of the distal segment (Fig. 1 B ), a large column of Sudan-black-colored castor oil wasinjected into the proximal tubule through barrel C toprevent downstream flow of native tubular fluid. The oil column wassubsequently split by injection of the control solution at a ratesufficient to lower distal tubular K concentration, asrecorded by the electrode, to the level of the perfusion fluid. Aftercontrol measurements, the experimental solution was injected through asingle-barreled micropipette (P) inserted into either a late proximalloop or the early distal tubule (Fig. 1 C ), and recordingswere made again. In a typical experiment, each nephron was perfused twoto three times with both an experimental and control solution, allowingpaired measurement of K secretion. In each animal, one tothree nephrons were perfused in the manner described.

Fig. 1. Schematic drawing of the stationary microperfusionmethod. A : a superficial segment of a proximal convolutedtubule is punctured with a double-barreled micropipette containingSudan-black colored castor oil (C) and control perfusate (P). B : a late superficial segment of the distal tubule ispunctured with a double-barreled microelectrode, consisting of apotassium-selective barrel (IE) and a reference barrel (Ref) connectedto a high-impedance electrometer (E). C : a single-barreledmicropipette containing the experimental solution (S) is inserted intoa late segment of the proximal tubule or, as shown, the early distaltubule.

Solutions and chemicals. The composition of the solutions used in the present study arepresented in Table 1. It should be notedthat the control solution (100 mM NaCl) was bicarbonate free and usedat either pH 7.0 or 8.0. In all of the other solutions, NaCl, reducedto 20 or 2 mM, was replaced by sodium salts of acetate, sulfate, orbicarbonate. Acetate was used because it exerted a similar effect tothat of sulfate and has a soluble barium salt. Raffinose pentahydrate(Riedel-de Haën, Hannover, Germany) was added to each solution toestablish isosmolality with plasma. Under these equilibrium conditions,some Na reabsorption may persist but fluid reabsorption isminimized so that increases in luminal K concentrationrelate directly to K secretion. Furosemide and[(dihydroindenyl)oxy]alkanoic acid (DIOA), both establishedinhibitors of K -Cl cotransport ( 7, 29 ), were dissolved in dimethylsulfoxide before dilution;vehicle alone (0.5%) was included in the control solutions.Hexamethyldisilazane and K ionophore cocktail A were both obtained fromFluka (Buchs, Switzerland). All other reagents were from Sigma.
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Table 1. Perfusion solutions& t# \3 h7 ~& u) n
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Ion-selective microelectrodes. Double-barreled microelectrodes were pulled from assymetricborosilicate glass to a tip diameter of ~1 µm. The smaller,reference barrel was backfilled with a solution containing 0.24 M NaCland 0.76 M Na acetate, colored by FD&C green. The total cation/anion mobility of this solution was similar because the mobility in asolution of Cl is larger than that of Na  ,whereas that of acetate is smaller. The animal was grounded (tail), andmeasurements of transepithelial potential difference were made throughthe reference barrel.0 P& s% C- l+ W8 L; b8 G

The larger barrel was exposed for 30 min at room temperature tohexamethyldisilazane before the tip was filled with K ionophore. Silanized electrodes were kept in a desiccator until the dayof the experiment, when they were filled with the electrolyte solutions( 1 ). The electrode was calibrated before and after eachimpalement by superfusion onto the kidney surface of standards (kept at37°C). Standards of 3, 10, and 30 mM KCl were used, and 100 mM NaClwas added to each standard to compensate for the fact that theK ionophore has an inherent sensitivity to sodium. Themean voltage difference per decade change in K concentration was similar to values previously reported by us ( 1 ). The voltage difference between the reference andion-selective barrels, representing luminal K activity,was recorded using a differential, high-impedance electrometer (model223, WPI, New Haven, CT) and sampled every second by an analog-digitalconverter (Lynx, São Paulo, Brazil).3 ?! B: h) ^+ x; H! g' X

Analysis and statistics. For each perfusion, luminal K activity increased from theinitial value of 0.5 mM, given as [K ] o, to astationary K concentration([K ] s ) as a result of K secretion into the nephron. These data were analyzed by aVisual Basic program that fit an exponential to the approach of luminal K activities to the stationary level, i.e., by plottingthe increase in luminal K activity against time. Thehalf-time ( t 1/2 ) of the approach of K activity to the stationary level was calculated fromthis exponential, and secretory K fluxes( J K ) were obtained by the followingrelationship, where r is tubule radius and other terms areas defined above K  (nmol·cm −2 ·s −1 ) =  ln 2 t ½ ·([K + ] s  − [K + ] o )· r 29 D1 _8 m! [5 m. l* P* ~& R7 ~

A value for each variable was obtained from individualperfusions: the mean of repeated perfusions under each condition was calculated to give a pair of values for each tubule, and n corresponds to the number of tubules perfused. Statisticalcomparisons were made using Student's paired t -test or,when nonpaired groups were compared, analysis of variance and theBonferroni contrast test. The probability of 0.05 was taken as thelimit of statistical significance.2 b8 S. q  ^5 W* a

RESULTS: H1 R. M' m2 O5 R
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A representative graph plotting the approach of the luminalK concentration to the stationary value (i.e.,K s luminal K concentration)against time is shown in Fig. 2.Figure 2 A details the secretory response under controlconditions (100 mM NaCl). Similar data obtained with a solution inwhich Cl was largely replaced by sulfate is depicted inFig. 2 B. Data were obtained from the same superficial distaltubule, and paired data sets were acquired for each setof experimental conditions. These curves were used to calculate t 1/2, an index of the rate of K secretion. According to the equation cited in METHODS, J K depends on [K ] s and on the rate of the approach to [K ] s fromthe initial perfusion value of 0.5 mM (i.e.,[K ] o ), given asln2 /t 1/2. From an inspection of Table 2, it is apparent that paired values for[K ] s were similar for Cl replacement with acetate, sulfate, or bicarbonate. Modulation ofK secretion by acute changes in luminal components thusreflects increases or decreases in t 1/2. It isapparent that the rate at which the steady-state luminal K concentration is established is greater, and t 1/2 is smaller, when Cl isreplaced by a less permeant anionic species.1 u! o, i+ q- I$ S# }3 d
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Fig. 2. Approach of luminal K concentration([K ]) to steady-state level([K ] s ) from the initial perfusateconcentration of 0.5 mM ([K ] o ). The changein [K ], which equals[K ] s [K ] at time t, is plotted as a function of time (s).,Experimental points; solid lines, exponentials fitted to data. A : control perfusion (100 mM NaCl). B : perfusionof the same nephron segment using a solution in which 80 mMCl has been replaced by sulfate., r& i: t, Y7 A& ~2 V
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Table 2. Stationary potassium concentration and approach to half-time duringpaired perfusion of the late distal tubule
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Calculation of J K from[K ] s [K ] o and t 1/2 yields the data presented in Figs. 3-6.A significant increase in J K was observed wheneither sulfate or acetate replaced luminal Cl. When theprincipal anion was bicarbonate, a further increment was recorded (Fig. 3 ). As shown in Table 3, perfusion withsulfate-containing solutions resulted in only a minor increase in thelumen-negative transepithelial potential difference, whereas neitherbicarbonate nor acetate altered this difference. These data indicatethat the role of changes in transepithelial potential difference in mediating K secretion is minor.

Fig. 3. Net K secretory flux( J K ) calculated from[K ] s and t 1/2,during paired perfusion of the distal tubule with control solutions(Cl ) and solutions in which Cl has beenreplaced by acetate, sulfate, or bicarbonate, as detailed in Table 1.Values are means ± SE; n = 9, 14, and 18 tubules,respectively. * P P t -test. The effect of bicarbonate was significantlygreater than that of either acetate or sulfate ( P t -test).
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Fig. 4. Net J K during paired perfusion ofCl and acetate and of acetate plus barium (Ba) andacetate plus barium plus furosemide (furo). Ba (3 mM) reducedacetate-stimulated J K by ~80%( P P n = 14 tubules for each paired condition. * P P." s  ^1 U: j3 S) S' Z) H
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Fig. 5. Top : J K increases asluminal Cl concentration decreases from 100 ( P P Bottom : stimulatory effect of reducing luminalCl concentration is abolished by either furosemide (1 mM; P P.2 p, ?& r( i: ~/ ]

Fig. 6. K secretion in the distal tubule duringluminal perfusion with the control perfusate (100 mM NaCl) at pH 7.0 and 8.0. Values are means ± SE; n = 11 (pH) and18 (bicarbonate) tubules. Increasing the pH of the luminal fluidsignificantly increased J K (* P P
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Table 3. Transepithelial potential difference in the late distal tubule duringperfusion with solutions of differing principal anions& b4 n1 k: ~2 V) ?0 V$ _

To assess the role of channels in mediating K secretion,Ba 2  was added to an acetate-containing perfusate.Ba 2  markedly reduced J K (Fig. 4 ),leaving a residual K secretion of 0.18 nmol · cm 2 · s 1. Theremaining J K was of a similar magnitude to theincrement in K secretion resulting from replacement ofCl with acetate (0.23 nmol · cm 2 · s 1 ),suggesting that the effect of acetate was not mediated through ionchannels. Addition of furosemide (1 mM) to the solution further reducedK secretion (Fig. 4 ). The remainder was stillsignificantly greater than zero ( P flux of K drivenalong the favorable electrochemical gradient ( 42.0 ± 1.7 mV)., K- B  T0 i4 H4 D' t0 |

Taken together, these observations strongly suggest that electroneutralK -Cl cotransport is operative after theestablishment of a favorable cell-to-lumen Cl concentration gradient. This hypothesis is supported by the observation that increasing the driving force for K -Cl cotransport by reducing luminal Cl concentration from 20 to 2 mM evoked a further increase in K secretion (Fig. 5, top ). With conditions employed herein, a reduction inluminal Cl is associated with an increase in luminalsulfate, and we cannot preclude a direct effect of the replacementanion. Nevertheless, it is noteworthy that either furosemide or DIOA (amore selective inhibitor of K -Cl cotransport) was able to reduce anion/low-Cl -stimulatedK secretion to levels observed with 100 mMCl (Fig. 5, bottom ). In addition, in thepresence of 100 mM Cl in the lumen, 100 µM DIOA did notaffect K secretion significantly. These experiments thusimply that the magnitude of the cell-to-luminal Cl concentration gradient rather than the nature of the luminal anionaccounts for the augmentation of K secretion.
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In a series of further experiments, we investigated the effect ofchanges in luminal pH on K secretion because the effectsof bicarbonate could be attributed to alkalinization of the tubularfluid. Increasing pH of the control (100 mM NaCl) solution from 7.0 to8.0 resulted in a significant stimulation of K secretion(Fig. 6 ). However, this increment was significantly less than thatresulting from perfusion of the lumen with a bicarbonate solution, alsoat pH 8.0. It is notable that the increment in J K resulting from alkalinzation of the tubularfluid was similar in magnitude to the difference between the respectivestimulations resulting from bicarbonate and from sulfate. To furtherinvestigate the mechanism by which alkalinization of the luminalperfusate enhanced K secretion, we performed theexperiments depicted in Fig. 7.Increasing the pH of the acetate-containing perfusate from 7.0 to 8.2 resulted in a marked increase in K secretion, despite thepresence of Ba 2  in both solutions.' l  W4 P, A" p! W' Z
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Fig. 7. Increasing the pH of the acetate perfusate from 7.0 to8.2 stimulates J K despite the continued presenceof Ba (3 mM). Values are means ± SE; n = 16 tubules; **= P

DISCUSSION/ [8 o1 E4 {& y6 N5 w
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K secretion in the distal nephron proceeds by twodistinct processes: active uptake of K in exchange forNa by the Na -K -ATPase across thebasolateral membrane provides the driving force for translocationacross the apical membrane along a favorable electrochemical gradient.The present investigation concerns the mechanisms by which changes inthe composition of the tubular fluid, independently of maneuvers thatalter the peritubular fluid, affect K secretion. We havespecifically focused on the mechanisms by which changes in theconcentration of distal tubular bicarbonate and other anions modulateK transport.

Previous studies have firmly established several luminal factors knownto alter K secretion in the distal convoluted tubule andinitial and final collecting ducts. These factors include the deliveryof Na ( 12 ), fluid ( 13 ), andCa 2  ( 21 ), the pH of fluid ( 3 ),and the nature of the principal anionic species ( 2, 26, 27 ). Studies in humans have defined the important role ofbicarbonate as a powerful stimulus of K secretion( 4, 15, 16 ) and have shown that its kaliuretic potencyexceeds that of other poorly reabsorbable anions such as sulfate./ h$ I' x& S, w; `7 v
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In the present study, K secretion in the initialcollecting duct was measured under equilibrium conditions in whichNa reabsorption was not associated with significant fluidmovement. The main findings were 1 ) replacement ofCl with either sulfate or acetate increased the rate ofK secretion; 2 ) replacement of Cl with bicarbonate elicited a further increment in K secretion; and 3 ) increasing the pH of the control perfusate stimulated K secretion.

The effect of sulfate and acetate. It has been suggested that impermeant anions increase the luminalnegativity of the transepithelial potential difference, therebyimposing a more favorable electrochemical gradient for K secretion ( 5, 11 ). That the transepithelial potentialdifference regulates K secretion is beyond doubt( 9 ). The importance of this variable in the present study,however, must be questioned because sulfate induced only a smallincrease in the lumen-negative transepithelial potential difference andboth acetate and bicarbonate were without effect. Our data imply that achange in potential difference would contribute in only a minor way tothe anion-stimulated K secretion and are in accord withearlier studies which found that sulfate can stimulate K secretion even when the negative transepithelial potential difference is abolished ( 27 ).
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K channels andK -Cl cotransport. It is reasonable to conclude that a large fraction of K secretion in our experiments was mediated by diffusion across the apical membrane through K channels because addition to thetubular fluid of Ba 2 , an effective blocker of both thesmall-conductance and the maxi-K channel ( 10 ), reduced J K by ~80% (Figs. 4 and 7 ). However, the persistence of a residual J K suggests additionalsecretory pathways. It is possible that K -Cl cotransport participated in distal nephron K secretion asfirst proposed on the basis of experiments demonstrating thatK secretion was stimulated by low concentrations ofluminal Cl ( 27 ), even in the presence ofBa 2  ( 6 ). Ba 2  -insensitiveK secretion has also been reported in the isolatedcortical collecting duct ( 20, 24 ) but only in animalstreated with deoxycorticosterone acetate.
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The present study confirmed the presence of aBa 2  -insensitive component of K secretion inthe distal nephron. This component was almost completely inhibited byboth furosemide and DIOA (Figs. 4 and 5 ). Similarly, maneuvers designedto increase the cell-to-lumen Cl concentration gradientevoked a gradient-dependent increase in J K, anincrease that was eradicated by K -Cl cotransport inhibition (Fig. 5 ). Although the specificity of K -Cl cotransport inhibitors is limited, themost likely interpretation of these findings is thatK -Cl cotransport contributes to thestimulation of J K by acetate, sulfate, andbicarbonate. Ba 2  -insensitive K secretion inthe late distal tubule of control rats had previously been demonstrated( 1 ) and supports the possibility thatK -Cl cotransport is operative under normalconditions. In support of this hypothesis, addition of eitherfurosemide or DIOA to low- Cl perfusates reducedK secretion to a level that was slightly below that foundwith 100 mM Cl. However, we observed that addition ofDIOA to 100 mM Cl -containing perfusates did not causesignificant alteration of K transport, suggesting thatK -Cl cotransport is only operative when thecell-to-lumen gradient is favorable (Fig. 5 ). The observation of acomponent of Ba 2  -insensitive K secretion athigher luminal or urinary Cl concentrations ( 4, 15, 20, 24 ) suggests the possibility of K secretion viaa paracellular transport route.
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Effect of bicarbonate and pH. K -Cl cotransport would be expected tocontribute to the stimulation of J K resultingfrom the replacement of Cl with bicarbonate (Fig. 3 ) forthe reasons cited above. Nevertheless, bicarbonate has additionaleffects associated with the alkaline pH of such solutions. There is astrong correlation between the pH of the tubular fluid andK secretion in the distal nephron ( 17, 18 ),and reducing the perfusate pH from 7.4 to 6.8 impairs K secretion in the rabbit cortical collecting duct ( 3 ). Inthe present study, we found that increasing the pH of the control solution (100 mM NaCl) stimulated distal K secretion.Although the effect of cytoplasmic pH on K channelactivity has been repeatedly demonstrated ( 8, 18 ), severalobservations make it very unlikely that increasing luminal pH mayenhance secretion through the small-conductance K channelof the principal cell (ROMK). First, ROMK channels are insensitive tochanges in extracellular pH ( 8, 10, 33 ). Second,intracellular pH of the principal cell was shown to be unaffected bychanges in luminal pH due to the apparent lack of an acid-basetransporter in the apical membrane ( 30 ). Sensitivity ofROMK to pH seems thus to be limited to the cytosole ( 8, 18 ). Finally, addition of Ba 2  to the acetateperfusate did not ablate the stimulatory effect on J K of increasing pH from 7.0 to 8.2. Thissuggests that luminal pH augments K efflux through aBa 2  -insensitive pathway. Two possibilities should beconsidered. The first is that changes in luminal pH activateK -Cl cotransport. Because the cell-to-lumenCl concentration gradient remained constant duringexperiments, extracellular pH would have to exert a direct modulatingaction on K -Cl cotransport. An alternativehypothesis invokes a role for the TASK2 K channel, whichis activated by extracellular alkalinization over the physiologicalrange yet is relatively insensitive to Ba 2  block( 22 ). In situ hybridization studies localize TASK2expression to the cortical distal tubules and collecting ducts, but aclearly defined role for these channels is lacking.
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Taken together, our data suggest an important role ofK -Cl cotransport in mediating anion effectson K secretion. Because distal K secretionhas been attributed mostly to the principal cell of cortical collectingduct, we have incorporated K -Cl cotransportinto this cell. This suggestion is shown in our model of K secretion in the distal nephron (Fig. 8 A ). The molecular nature ofthis transport is unclear, although three of the four known isoforms ofthe K -Cl cotransporter are present in thekidney: KCC3 and KCC4 have been found in renal proximal tubules,particularly in the basolateral membrane ( 19 ). KCC1 isexpressed in the apical membrane of the thick ascending limb; morerecent evidence localizes this transporter also to distal tubule andcollecting duct (Mount DB, personal communication). Nevertheless,alternative explanations for our findings should be considered (Fig. 8 B ). Stimulation of J K by both thehigh luminal pH and bicarbonate could also involve known transportmechanisms in B-intercalated cells. In these cells, coupling of theapically located Cl /bicarbonate exchanger ( 14, 23, 25, 28, 32 ) with H -K -ATPase has beensuggested to mediate Cl reabsorption ( 32 ).Under special circumstances, such coordinated activity could also beinvolved in KCl secretion. We consider the inversion of active KClreabsorption into secretion highly unlikely. However, alkalinization ofB-intercalated cells by reversal of Cl / bicarbonateexchange could reduce H -K -ATPase activity andthus diminish K reabsorption. In parallel with continuedK secretion via principal cells, enhanced net secretion ofK would result. However, this hypothesis must beconsidered uncertain because the activity ofH -K -ATPase appears to be significant onlywhen these cells are acidified ( 31 ). This issue requiresfurther investigation, especially in view of the fact that intercalatedcells have been found to display significant heterogeneity with respectto expression of acid-base transporters ( 28, 31 ).  A2 k8 _) E0 p8 {- H

Fig. 8. Possible models of collecting duct K transport. Top : K secretion in the principalcell involves active uptake of K in exchange forNa by Na -K -ATPase across thebasolateral membrane, which provides the driving force fortranslocation across the apical membrane along a favorableelectrochemical gradient. Luminal K transport involvesK channels and K-Cl cotransport. Luminal modulators ofK secretion include the transepithelial potentialdifference, the concentration of luminal Cl andbicarbonate, and pH. pH affects K channels from thecytoplasmic side, and K-Cl cotransport from the lumen. Bottom : alternative model involving B-type intercalated cellwith apical H -K -ATPase andCl /HCO 3 − exchange. Modified according toZhou et al. ( 32 ). CCD, cortical collecting duct.
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In conclusion, our studies show that elevation of bicarbonate in distaltubules is uniquely potent in stimulating K secretion. Thedata suggest an important role of K -Cl cotransport or coordinated changes inH -K -ATPase activity coupled toHCO 3 − /Cl exchange. The present findingsalso suggest that from a physiological point of view the proposedtransport mechanism (K -Cl cotransport) wouldbe operative in low-Cl urines, found in patients withlow-salt diets, and particularly in conditions of increased bicarbonateexcretion such as metabolic alkalosis.
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ACKNOWLEDGEMENTS

M. A. Bailey was funded by the Wellcome Trust. G. Giebisch wassupported by National Institute of Diabetes and Digestive and KidneyDiseases Grant DK-17433. R. Musa-Aziz and G. Malnic were supported bythe Fundação de Amparo à Pesquisa do Estado de São Paulo and by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (Pronex).) w, M" r& e: o, X: ?
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* Z, C& [9 J; \( C

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5. Clapp, JR,Rector FC,andSeldin DW. Effect of unreabsorbed anion on proximal and distal transtubular potentials in the rat. Am J Physiol 202:781-786,1962 .
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7. Ellory, JC,Hall AC,Ody SO,Englert HC,Mania D,andLang HJ. Selective inhibitors of KCl cotransport in human red cells. FEBS Lett 262:215-218,1990  .
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