Basolateral Na+/H+ exchange maintains potassium secretion during diminished sodium transport in the rabbit cortical collecting duct
2008; Elsevier BV; Volume: 75; Issue: 1 Linguagem: Inglês
10.1038/ki.2008.447
ISSN1523-1755
AutoresShigeaki Muto, Shuichi Tsuruoka, Yukio Miyata, Akio Fujimura, Eiji Kusano, Wen‐Hui Wang, Donald W. Seldin, Gerhard Giebisch,
Tópico(s)Ion channel regulation and function
ResumoStimulation of the basolateral Na+/K+-ATPase in the isolated perfused rabbit cortical collecting duct by raising either bath potassium or lumen sodium increases potassium secretion, sodium absorption and their apical conductances. Here we determined the effect of stimulating Na+/K+-ATPase on potassium secretion without luminal sodium transport. Acutely raising bath potassium concentrations from 2.5 to 8.5 mM, without luminal sodium, depolarized the basolateral membrane and transepithelial voltages while increasing the transepithelial, basolateral and apical membrane conductances of principal cells. Fractional apical membrane resistance and cell pH were elevated. Net potassium secretion was maintained albeit diminished and was still enhanced by raising bath potassium, but was reduced by basolateral ethylisopropylamiloride, an inhibitor of Na+/H+ exchange. Luminal iberitoxin, a specific inhibitor of the calcium-activated big-conductance potassium (BK) channel, impaired potassium secretion both in the presence and absence of luminal sodium. In contrast, iberitoxin did not affect luminal sodium transport. We conclude that basolateral Na+/H+ exchange in the cortical collecting duct plays an important role in maintaining potassium secretion during compromised sodium supplies and that BK channels contribute to potassium secretion. Stimulation of the basolateral Na+/K+-ATPase in the isolated perfused rabbit cortical collecting duct by raising either bath potassium or lumen sodium increases potassium secretion, sodium absorption and their apical conductances. Here we determined the effect of stimulating Na+/K+-ATPase on potassium secretion without luminal sodium transport. Acutely raising bath potassium concentrations from 2.5 to 8.5 mM, without luminal sodium, depolarized the basolateral membrane and transepithelial voltages while increasing the transepithelial, basolateral and apical membrane conductances of principal cells. Fractional apical membrane resistance and cell pH were elevated. Net potassium secretion was maintained albeit diminished and was still enhanced by raising bath potassium, but was reduced by basolateral ethylisopropylamiloride, an inhibitor of Na+/H+ exchange. Luminal iberitoxin, a specific inhibitor of the calcium-activated big-conductance potassium (BK) channel, impaired potassium secretion both in the presence and absence of luminal sodium. In contrast, iberitoxin did not affect luminal sodium transport. We conclude that basolateral Na+/H+ exchange in the cortical collecting duct plays an important role in maintaining potassium secretion during compromised sodium supplies and that BK channels contribute to potassium secretion. K+ secretion in the cortical collecting duct (CCD) is tightly coupled to Na+ reabsorption. Increased Na+ entry across the apical membrane stimulates the Na+-K+-ATPase, which enhances Na+ extrusion and K+ uptake across the basolateral membrane.1.Malnic G. Muto S. Giebisch G. Regulation of potassium excretion.in: Alpern R.J. Hebert S.C. Seldin and Giebisch's The kidney: Physiology and pathophysiology. 4th edn. Elsevier, San Diego2007: 1301-1348Google Scholar This is followed by passive diffusion of K+ across the apical membrane along a favorable electrochemical gradient. We have previously demonstrated that in the isolated perfused rabbit CCD raising bath K+ from 2.5 to 8.5 mM in the presence of luminal Na+ increases K+ secretion and Na+ reabsorption as well as apical Na+ and K+ conductances.2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar, 3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar, 4.Muto S. Giebisch G. Sansom S. An acute increase of peritubular K stimulates K transport through cell pathways of CCT.Am J Physiol Renal Fluid Electrolyte Physiol. 1988; 255: F108-F114PubMed Google Scholar However, K+ excretion may not completely depend on apical Na+ entry. For example, rats maintained on a low-Na+ diet can increase renal K+ secretion in response to acute K+ loading.5.Peterson L.N. Wright F.S. Effect of sodium intake on renal potassium excretion.Am J Physiol Renal Fluid Electrolyte Physiol. 1977; 2: F225-F234Google Scholar Furthermore, a significant kaliuretic response after acute K+ loading has been observed in dogs receiving amiloride, which inhibits apical Na+ reabsorption in the distal nephron.6.Yeyati N.L. Etcheverry J.C. Adrogue H.J. Kaliuretic response to potassium loading in amiloride-treated dogs.Renal Physiol Biochem. 1990; 13: 190-199PubMed Google Scholar Thus, it is conceivable that the kidney maintains K+ secretion by a mechanism, which does not require apical Na+ entry in principal cells. The aim of the present study is to test the hypothesis that basolateral Na+ entry via Na+/H+ exchange (NHE) could sustain Na+-K+-ATPase activity and K+ secretion in the CCD during compromised luminal Na+ absorption. First we examined the electrical properties of principal cell in response to raising basolateral K+ in the absence of luminal Na+. As shown previously,2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar, 4.Muto S. Giebisch G. Sansom S. An acute increase of peritubular K stimulates K transport through cell pathways of CCT.Am J Physiol Renal Fluid Electrolyte Physiol. 1988; 255: F108-F114PubMed Google Scholar raising basolateral K+ from 2.5 to 8.5 mM in the presence of luminal Na+ induced hyperpolarization of both transepithelial voltage (VT) and basolateral membrane voltage (VB) followed by depolarization. The initial transient hyperpolarization was the result of stimulating Na+-K+-ATPase whereas the subsequent depolarization was due to alterations of apical Na+ and K+ conductances as well as basolateral K+ conductance. Figure 1 is a typical recording showing the effect of raising basolateral K+ concentration on VT and VB. In contrast to the condition in which luminal Na+ is present, the transient hyperpolarization of VT and VB was absent following raising basolateral K+ to 8.5 mM. Raising basolateral K+ depolarized VT from 1.0±0.4 to 8.3±0.5 mV and VB from -82.7±1.2 to -75.1±1.1 mV without significant changes of apical membrane voltage (VA; Table 1). Also, both transepithelial conductance (GT) and the fractional apical membrane resistance (fRA) significantly increased from 6.5±0.1 to 7.4±0.1 mS/cm2 and 0.56±0.01 to 0.60±0.01, respectively. It is possible that the expected transient hyperpolarization of VT and VB was masked by a rapid depolarization induced by changing the K+ equilibrium potential following raising basolateral K+. Alternatively, removal of luminal Na+ might weaken the stimulation of Na+-K+-ATPase induced by raising basolateral K+ concentration. The first possibility was confirmed by experiments in which the above experiment was conducted in the presence of Ba2+ in the bath solution (Figure 1). It is apparent that raising bath K+ concentration caused a transient hyperpolarization of VT and VB. Thus, raising K+ concentration could still activate Na+-K+-ATPase in the absence of luminal Na+. Moreover, in the absence of luminal Na+, stimulation of Na+-K+-ATPase with 8.5 mM K+ increased both basolateral membrane conductance (GB) (from 11.7±0.9 to 15.7±1.4 mS/cm2; n=18, P<0.001) and apical membrane conductance (GA) (from 8.4±0.5 to 9.2±0.8 mS/cm2; n=18, P<0.05; Figure 2). Because removal of luminal Na+ is expected to abolish the apical Na+ entry, the observed increase in the GA is best explained by augmentation of the apical K+ conductance. This view is also supported by the observation that adding amiloride to the lumen did not affect, whereas inhibition of apical K+ conductance with luminal Ba2+ did abolish the high bath-K+-induced changes in fRA (data not shown).Table 1Effects of raising bath K+ from 2.5 to 8.5 mM in the absence of luminal Na+ on barrier voltages and conductances in the CCDsBath K (mM)2.58.52.5 (recover)VT, mV (n=53)1.0±0.48.3±0.5*0.8±0.4VB, mV (n=53)-82.7±1.2-75.1±1.1*-82.9±1.3VA, mV (n=53)83.8±1.283.4±1.283.7±1.3GT, mS/cm2 (n=53)6.5±0.17.4±0.1*6.3±0.1fRA (n=53)0.56±0.010.60±0.01*0.56±0.02Values are mean±s.e. Data at 8.5 mM K+ in the bath were taken at the peak of the depolarization.*P<0.001 compared with preceding period. Open table in a new tab Figure 2Bar graph summarizing effects of raising bath K+ from 2.5 to 8.5 mM on the basolateral membrane conductance (GB), apical membrane conductance (GA), and tight junction conductance (GTJ). *P<0.05 and **P<0.001 compared with 2.5 mM bath K+ (2.5K). The number of tubules examined is 18.View Large Image Figure ViewerDownload (PPT) Values are mean±s.e. Data at 8.5 mM K+ in the bath were taken at the peak of the depolarization. *P<0.001 compared with preceding period. To evaluate the role of Na+-K+-ATPase in stimulating K+ secretion in the absence of luminal Na+, we examined the effect of raising bath K+ from 2.5 to 8.5 mM on net K+ secretion (JK). As shown in Figure 3, we confirmed that in the presence of luminal Na+, raising bath K+ from 2.5 to 8.5 mM stimulated JK from -12.1±0.8 to -19.3±0.6 peq/mm/min (n=6, P<0.001). Removal of luminal Na+ significantly attenuated net K+ secretion at both basolateral 2.5 and 8.5 mM K+ concentrations. However, raising bath K+ from 2.5 to 8.5 mM still increased JK from -6.5±0.2 to -8.5±0.1 peq/mm/min (n=4, P<0.001; Figure 3). Thus, removal of luminal Na+ did attenuate but not abolish the stimulatory effect of high bath K+ on net K+ secretion. Because raising bath K+ had no effect on the tight junction conductance (GTj; Figure 2), it is unlikely that an increase in JK upon raising bath K+ is due to back-leak through the paracellular pathway in the absence of luminal Na+. To test whether basolateral Na+ could be an alternative source of Na+ for Na+-K+-ATPase when luminal Na+ transport is compromised, we hypothesized that Na+ recycles across the basolateral membrane by NHE. We used pH-sensitive dye to measure intracellular pH (pHi) during changes in basolateral K+ in the absence of luminal Na+. Figure 4 summarizes data obtained in a typical experiment. It is apparent that raising bath K+ significantly increased pHi from 7.36±0.04 to 7.41±0.04 (n=14, P<0.01). The addition of ethylisopropylamiloride (EIPA) to the bath significantly decreased pHi from 7.38±0.05 to 7.30±0.05 (n=8, P<0.005). It is also apparent that high bath K+ had no effect on pHi (n=8, 7.30±0.05 vs 7.29±0.05) after inhibition of NHE. Thus, in the absence of luminal Na+, NHE plays a significant role in sustaining the activity of basolateral Na+-K+-ATPase. Basolateral K+ channels are sensitive to pH and could be affected by inhibition of NHE. We compared the effects of raising bath K+ on electrical properties of principal cells in the absence and presence of bath EIPA without luminal Na+. As expected, raising bath K+ significantly depolarized both VT and VB. Addition of bath EIPA significantly depolarized VT from 0.7±1.0 to 5.4±1.2 mV (n=15, P<0.001) and VB from -83.5±2.3 to -76.4±2.5 mV (n=15, P<0.001; Figure 5). Moreover, the effect of changing bath K+ on both VT (Δ=4.5±0.3 vs 8.9±0.7 mV, n=15, P<0.001) and VB (Δ=5.2±0.3 vs 9.8±0.5 mV, n=15, P<0.001) in the presence of bath EIPA was significantly diminished compared with that in its absence. These data support the participation of NHE in modulating basolateral K+ channel activity. To further explore the role of basolateral NHE in the regulation of K+ secretion in the absence of luminal Na+, we measured JK in the presence of bath 100 μM EIPA (see Figure 6). Inhibition of basolateral NHE significantly decreased JK with both 2.5 mM (from -6.5±0.2 (n=4) to -3.5±0.2 peq/mm/min (n=6), P<0.001) and 8.5 mM K+ in the bath (from -8.5±0.8 (n=4) to -4.6±0.2 peq/mm/min (n=6), P<0.001). In contrast, these EIPA effects were absent in the presence of luminal Na+, suggesting a role of basolateral NHE in K+ secretion only when apical Na+ supply is compromised. Both the renal outer medullary K+ channel (ROMK) and the Ca2+-activated big-conductance K+ (BK) channel have been implicated in renal K+ secretion.1.Malnic G. Muto S. Giebisch G. Regulation of potassium excretion.in: Alpern R.J. Hebert S.C. Seldin and Giebisch's The kidney: Physiology and pathophysiology. 4th edn. Elsevier, San Diego2007: 1301-1348Google Scholar, 7.Bailey M.A. Cantone A. Yan Q. et al.Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of type II Bartter's syndrome and in adaptation to a high-K diet.Kidney Int. 2006; 70: 51-59Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 8.Wang W. Schwab A. Giebisch G. Regulation of small-conductance K+ channel in apical membrane of rat cortical collecting tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 1990; 259: F494-F502PubMed Google Scholar In the present study, we examined whether BK channels mediate K+ secretion in response to high bath K+. We tested the effect of iberiotoxin (IBX), a specific inhibitor of BK channels, on JK under control conditions (luminal Na+) and in the absence of luminal Na+. As shown in Figure 7, in the presence of luminal Na+, inhibition of apical BK channels decreased JK from -12.1±0.8 (n=6) to -4.1±0.3 peq/mm/min (n=4, P<0.001) in the 2.5 mM K+ bath and from -19.3±0.6 (n=6) to -8.5±0.4 peq/mm/min (n=4, P<0.001) in the 8.5 mM K+ bath. In the absence of luminal Na+, IBX decreased JK from -6.5±0.2 (n=4) to -2.3±0.2 peq/mm/min (n=4, P<0.001) in the 2.5 mM K+ bath and -8.5±0.4 to -3.6±0.1 peq/mm/min (n=4, P<0.001) in the 8.5 mM K+ bath. Thus, BK channels are involved in mediating K+ secretion regardless of the presence of luminal Na+. The main findings of the present study are the maintenance of collecting duct K+ secretion in the absence of significant luminal Na+ transport and the activation of apical BK channels with stimulation of basolateral Na+-K+-ATPase. The K+ concentration of 8.5 mM to stimulate Na+-K+-ATPase is not a physiological K+ concentration. The main reason for choosing such a high K+ level was to test the effect of maximal stimulation of Na+-K+-ATPase on Na+ and K+ transport. When luminal Na+ transport is compromised, it is expected that K+ secretion would be impaired and that plasma K+ could increase. As a consequence, Na+-K+-ATPase would be stimulated to enhance K+ uptake across the basolateral membrane, leading to stimulation of K+ secretion. Luminal Na+ transport plays an important role in K+ secretion in the CCD. First, diffusion of Na+ provides an important driving force for K+ secretion through depolarization of the apical membrane. Second, apical entry of Na+ stimulates the basolateral Na+-K+-ATPase, which plays a key role for K+ entry across the basolateral membrane. Accordingly, it would be expected that the absence of luminal Na+ transport would decrease K+ secretion by curtailing the supply of Na+ for Na+-K+-ATPase activity. However, the present observation that removal of luminal Na+ suppressed but did not abolish K+ secretion in the CCD strongly suggests the presence of an alternative mechanism by which Na+-K+-ATPase continues operating in the absence of luminal Na+ transport. We suggest that the NHE, which is present in the basolateral membrane of the rabbit CCD9.Sauer M. Flemmer A. Thurau K. et al.Sodium entry routes in principal and intercalated cells of the isolated perfused cortical collecting duct.Pfluegers Arch. 1990; 416: 88-93Crossref PubMed Scopus (20) Google Scholar and regulates intracellular pH,10.Chaillet J.R. Lopes A.G. Boron W.F. Basolateral Na-H exchange in the rabbit cortical collecting tubule.J Gen Physiol. 1985; 86: 795-812Crossref PubMed Scopus (78) Google Scholar is responsible for providing Na+ for the Na+-K+-ATPase. This hypothesis is supported by the observation that inhibition of the NHE significantly diminished K+ secretion in the absence of luminal Na+. In contrast, when Na+ is present in the lumen, such basolateral NHE is not expected to play a major role in providing Na+ for Na+-K+-ATPase. This conclusion is supported by the finding that in the presence of luminal Na+ entry, inhibition of NHE had no effect on K+ secretion. Thus, it is possible that the basolateral NHE becomes a major Na+ provider for the Na+-K+-ATPase only when the luminal Na+ transport is compromised. In addition to NHE, other basolateral Na+ entry mechanisms may also be involved in maintaining Na+-K+-ATPase activity. This notion is supported by the finding that EIPA did not completely block the high bath K+-induced increase in JK in the absence of luminal Na+ entry. Thus, other basolateral Na+ entry pathways than NHE, including 3Na+/1Ca2+ exchange,11.Taniguchi S. Marchetti J. Morel F. Na/Ca exchangers in collecting cells of rat kidney.Pfluegers Arch. 1989; 415: 191-197Crossref PubMed Scopus (24) Google Scholar may also be involved in the high bath K+-induced increase in JK. The physiological relevance of the present study is illustrated by a report that Yanomamo Indians, who consume a nominally Na+ free and high K+ diet, are able to secrete 200 mEq of K+ per 24 h while their Na+ excretion was only 1 mEq.12.Oliver W.J. Cohen E.L. Neel J.V. Blood pressure, sodium intake, and sodium related hormones in the Yanomamo Indians, a 'no-salt' culture.Circulation. 1975; 52: 146-151Crossref PubMed Scopus (363) Google Scholar Under such conditions, Na+ in distal nephron segments must have declined sharply and basolateral Na+ entry could be responsible for sustaining K+ secretion without significant Na+ absorption. It has also been shown that amiloride-treated dogs continue enhanced K+ excretion in response to K+ loading.6.Yeyati N.L. Etcheverry J.C. Adrogue H.J. Kaliuretic response to potassium loading in amiloride-treated dogs.Renal Physiol Biochem. 1990; 13: 190-199PubMed Google Scholar The second finding of the present study is that BK channels are involved in K+ secretion in the CCD. Two types of K+ channels are expressed in the apical membrane of connecting tubule and the CCD.8.Wang W. Schwab A. Giebisch G. Regulation of small-conductance K+ channel in apical membrane of rat cortical collecting tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 1990; 259: F494-F502PubMed Google Scholar, 13.Frindt G. Palmer L.G. Low-conductance K channel in apical membrane of rat cortical collecting tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 1989; 256: F143-F151PubMed Google Scholar, 14.Hirsch J. Leipziger J. Frobe U. et al.Regulation and possible physiological role of Ca2+-dependent K+ channel of cortical collecting ducts of rat.Pfluegers Arch. 1993; 422: 492-498Crossref PubMed Scopus (71) Google Scholar It is well established that ROMKs are mainly responsible for K+ secretion under physiological conditions.8.Wang W. Schwab A. Giebisch G. Regulation of small-conductance K+ channel in apical membrane of rat cortical collecting tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 1990; 259: F494-F502PubMed Google Scholar, 13.Frindt G. Palmer L.G. Low-conductance K channel in apical membrane of rat cortical collecting tubule.Am J Physiol Renal Fluid Electrolyte Physiol. 1989; 256: F143-F151PubMed Google Scholar However, BK channels are involved in mediating K+ secretion in the distal nephron when the tubular flow rate is high or dietary K+ intake increases.7.Bailey M.A. Cantone A. Yan Q. et al.Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of type II Bartter's syndrome and in adaptation to a high-K diet.Kidney Int. 2006; 70: 51-59Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 15.Liu W. Morimoto T. Woda C. et al.Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting duct.Am J Physiol Renal Physiol. 2007; 293: F227-F235Crossref PubMed Scopus (73) Google Scholar, 16.Woda C.B. Bragin A. Kleyman T.R. et al.Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel.Am J Physiol Renal Physiol. 2001; 280: F786-F793PubMed Google Scholar, 17.Woda C.B. Miyawaki N. Ramalakshmi S. et al.Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting duct: functional and molecular aspects.Am J Physiol Renal Physiol. 2003; 285: F629-F639Crossref PubMed Scopus (82) Google Scholar We confirmed the previous finding that BK channels are involved in K+ secretion in the CCD in the present experimental setting when tubular flow rate is high. Similar findings have been reported by Woda et al.17.Woda C.B. Miyawaki N. Ramalakshmi S. et al.Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting duct: functional and molecular aspects.Am J Physiol Renal Physiol. 2003; 285: F629-F639Crossref PubMed Scopus (82) Google Scholar in which inhibition of BK channel decreased JK in the rabbit CCD. However, we have demonstrated that BK channel-mediated K+ secretion could also be observed in the absence of luminal Na+ transport. However, if tubular flow rates were decreased to the low nanoliter range, inhibition of BK channels might have been less effective.17.Woda C.B. Miyawaki N. Ramalakshmi S. et al.Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting duct: functional and molecular aspects.Am J Physiol Renal Physiol. 2003; 285: F629-F639Crossref PubMed Scopus (82) Google Scholar Also, BK-dependent K+ secretion induced by stimulating Na+-K+-ATPase is unlikely due to increasing intracellular Ca2+ in response to raising bath K+ to 8.5 mM. Our previous study3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar has shown that acutely raising K+ to 8.5 mM significantly decreased intracellular Na+, presumably as a result of stimulation of Na+-K+-ATPase. As a consequence, intracellular Ca2+ falls in the presence of luminal 146.8 mM Na+. However, the effect of high bath K+ on intracellular Na+ and Ca2+ was significantly blunted in the presence of low luminal Na+ concentration (14.0 mM). Thus, we speculate that raising bath K+ should have a minimal effect on intracellular Ca2+ in the absence of luminal Na+. We believe that an alternative explanation accounting for the increase of intracellular Ca2+ required for activating BK channels is flow-induced Ca2+ influx, as shown by Woda et al.17.Woda C.B. Miyawaki N. Ramalakshmi S. et al.Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting duct: functional and molecular aspects.Am J Physiol Renal Physiol. 2003; 285: F629-F639Crossref PubMed Scopus (82) Google Scholar Figure 8 is a model illustrating the K+ secretory mechanism in the presence or absence of luminal Na+ at high tubular flow rates. In the presence of luminal Na+, Na+ entry via ENaC is the main source of Na+ for Na+-K+-ATPase and NHE activity does not play a significant role in maintaining Na+-K+-ATPase activity and K+ secretion. In contrast, when the luminal Na+ transport is compromised, NHE and other basolateral Na+ entry mechanisms may play a major role for sustaining Na+-K+-ATPase and K+ secretion via Na+ recycling across the basolateral membrane. We speculate that transit receptor potential channels (TRPC) may play a role in sustaining the electric current flow across the apical membrane. Their presence has been reported in the apical membrane of rabbit CCD and they could serve as a route for cation entry such as Ca2+ across the apical membrane.18.Taniguchi J. Tsuruoka S. Mizuno A. et al.TRPV4 as a flow sensor in flow-dependent K+ secretion from the cortical collecting duct.Am J Physiol Renal Physiol. 2007; 292: F667-F673Crossref PubMed Scopus (75) Google Scholar Such influx of cations should depolarize the apical membrane and thus provide a driving force for K+ secretion. Moreover, an increase in cell Ca2+ could stimulate Na+/Ca2+ exchange across the basolateral membrane and also provide an additional source of Na+ for the Na+-K+-ATPase. In our previous study,2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar the importance of a back-up system to forestall hyperkalemia under circumstances of sharply reduced distal Na+ delivery was described. High bath K+ was demonstrated to activate the basolateral electrogenic Na+-K+-ATPase, thereby ensuring adequate apical K+ secretion despite a constraint on apical Na+ entry. This identified plasma K+ as a Na+-independent regulator of K+ clearance. The present study discloses that Na+, no longer available through apical entry, can nevertheless be provided, at least part, by activation of basolateral NHE. It is low intracellular Na+, thereby providing the critical stimulus for basolateral Na+ entry necessary to sustain activity of the basolateral Na+-K+-ATPase. In conclusion, basolateral NHE plays an important role in maintaining Na+-K+-ATPase activity and K+ secretion when the luminal Na+ transport is compromised. At high tubular flow rates, BK channels are involved in mediating K+ secretion both in the presence and absence of luminal Na+. Animal protocols were approved by the Animal Experimental Committee at the Jichi Medical University. Female Japanese white rabbits (1.5–2.5 kg; Clea Japan, Tokyo, Japan) were maintained on a standard rabbit chow and tap water ad libitum. They were anesthetized with intravenous sodium pentobarbital (35 mg/kg), and both kidneys were removed. Thin slices (1–2 mm) were cut from the coronal section of each kidney and transferred to a dish containing dissecting solution composed of (in mM): 14 KCl, 44 K2HPO4, 14 KH2PO4, 9 NaHCO3, and 160 sucrose, a medium that had been shown to improve the quality of the kidney tissue.2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar, 3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar, 19.Muto S. Miyata Y. Imai M. et al.Troglitazone stimulates basolateral rheogenic Na+/HCO3- cotransport activity in rabbit proximal tubule S2 segments.Exp Nephrol. 2001; 9: 191-197Crossref PubMed Google Scholar, 20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar, 21.Muto S. Yasoshima K. Yoshitomi K. et al.Electrophysiological identification of α- and β-intercalated cells and their distribution along the rabbit distal nephron segments.J Clin Invest. 1990; 86: 1829-1839Crossref PubMed Scopus (91) Google Scholar The CCD segments were microdissected, mounted on glass pipettes, and perfused in vitro in a rapid-exchange chamber at 37 °C as described previously.2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar, 3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar, 4.Muto S. Giebisch G. Sansom S. An acute increase of peritubular K stimulates K transport through cell pathways of CCT.Am J Physiol Renal Fluid Electrolyte Physiol. 1988; 255: F108-F114PubMed Google Scholar, 19.Muto S. Miyata Y. Imai M. et al.Troglitazone stimulates basolateral rheogenic Na+/HCO3- cotransport activity in rabbit proximal tubule S2 segments.Exp Nephrol. 2001; 9: 191-197Crossref PubMed Google Scholar, 20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar, 21.Muto S. Yasoshima K. Yoshitomi K. et al.Electrophysiological identification of α- and β-intercalated cells and their distribution along the rabbit distal nephron segments.J Clin Invest. 1990; 86: 1829-1839Crossref PubMed Scopus (91) Google Scholar The composition of the control Na+ perfusion solution used in this study was as follows (in mM): 110 NaCl, 5 KCl, 25 NaHCO3, 0.8 Na2HPO4, 0.2 NaH2PO4, 10 Na-acetate, 1.8 CaCl2, 1.0 MgCl2, 8.3 glucose, and 5 alanine. The perfusion solution without Na+ was made by removing 110 mM NaCl, 1.8 mM KCl, 25 mM NaHCO3, 0.8 mM Na2HPO4, 0.2 mM NaH2PO4, 10 mM Na-acetate from the control Na+ perfusion solution and by adding 120 mM choline-Cl, 25 mM choline-HCO3, 0.8 mM K2HPO4, and 0.2 mM KH2PO4 to the control solution. The bathing solution including 2.5 mM K+ was made by removing 2.5 mM KCl from the control Na+ perfusion solution and by adding 2.5 mM NaCl to the control solution. The bathing solution containing 8.5 mM K+ was made by removing 3.5 mM NaCl from the control Na+ perfusion solution and by adding 3.5 mM KCl to the control solution All the solutions had an osmolality between 285 and 295 mOsm/kg H2O, and were equilibrated with 95% O2/5% CO2 and adjusted to pH 7.4 at 37 °C. The transepithelial and cellular electrical potentials were measured using methods described previously.2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar, 3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar, 4.Muto S. Giebisch G. Sansom S. An acute increase of peritubular K stimulates K transport through cell pathways of CCT.Am J Physiol Renal Fluid Electrolyte Physiol. 1988; 255: F108-F114PubMed Google Scholar, 20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar, 21.Muto S. Yasoshima K. Yoshitomi K. et al.Electrophysiological identification of α- and β-intercalated cells and their distribution along the rabbit distal nephron segments.J Clin Invest. 1990; 86: 1829-1839Crossref PubMed Scopus (91) Google Scholar, 22.Muto S. Giebisch G. Sansom S. Effects of adrenalectomy on CCD: evidence for differential response of two cell types.Am J Physiol Renal Fluid Electrolyte Physiol. 1987; 253: F742-F752PubMed Google Scholar Briefly, the VT was measured via a perfusion pipette connected to a dual channel electrometer (Duo 773; W-P Instruments, Inc., Sarasota, FL, USA) with a 3 M KCl-3% agar bridge and a calomel half-cell electrode. The VB was measured with 0.5 M KCl-filled microelectrodes. Both VT and VB were referenced to the bath (0 mV) and were recorded on a four-pen chart recorder (R64; Rikadenki, Tokyo, Japan). The VA was calculated as VA=VT-VB. In this study, the tubular lumen was perfused at flow rates of 20 nl/min. The liquid junction potential induced by removing luminal Na+ was corrected with free-flowing 3 M KCl electrodes.3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar Cable analysis was used to calculate the GT and the fRA as described previously.2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar, 3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar, 20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar, 21.Muto S. Yasoshima K. Yoshitomi K. et al.Electrophysiological identification of α- and β-intercalated cells and their distribution along the rabbit distal nephron segments.J Clin Invest. 1990; 86: 1829-1839Crossref PubMed Scopus (91) Google Scholar, 22.Muto S. Giebisch G. Sansom S. Effects of adrenalectomy on CCD: evidence for differential response of two cell types.Am J Physiol Renal Fluid Electrolyte Physiol. 1987; 253: F742-F752PubMed Google Scholar, 23.Sansom S.C. O'Neil R.G. Effects of mineralocorticoids on transport properties of cortical collecting duct basolateral membrane.Am J Physiol Renal Fluid Electrolyte Physiol. 1986; 251: F743-F757PubMed Google Scholar, 24.Schlatter E. Schafer J.A. Electrophysiological studies in principal cells of rat cortical collecting tubules. ADH increases the apical membrane Na+-conductance.Pfluegers Arch. 1987; 409: 81-92Crossref PubMed Scopus (121) Google Scholar For estimation of the GT, constant-current pulses, 50 nA (300 ms in duration, 10-s intervals), was injected into the tubule lumen via the perfusion pipette. The fRA was estimated from the ratio of the voltage deflection across the apical membrane and the entire epithelium at the point of impalement. We also estimated GA, GB, and GTj by measuring RT and fRA in the absence and presence of lumen Ba2+ (2 mM) as previously described.2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar, 3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar, 22.Muto S. Giebisch G. Sansom S. Effects of adrenalectomy on CCD: evidence for differential response of two cell types.Am J Physiol Renal Fluid Electrolyte Physiol. 1987; 253: F742-F752PubMed Google Scholar, 23.Sansom S.C. O'Neil R.G. Effects of mineralocorticoids on transport properties of cortical collecting duct basolateral membrane.Am J Physiol Renal Fluid Electrolyte Physiol. 1986; 251: F743-F757PubMed Google Scholar Only principal cells in the CCD were impaled, using methods of identification as previously described.2.Muto S. Asano Y. Seldin D. et al.Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.Am J Physiol Renal Physiol. 1999; 276: F143-F158PubMed Google Scholar, 3.Muto S. Asano Y. Wang W.H. et al.Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F945-F954Crossref PubMed Scopus (12) Google Scholar, 21.Muto S. Yasoshima K. Yoshitomi K. et al.Electrophysiological identification of α- and β-intercalated cells and their distribution along the rabbit distal nephron segments.J Clin Invest. 1990; 86: 1829-1839Crossref PubMed Scopus (91) Google Scholar, 22.Muto S. Giebisch G. Sansom S. Effects of adrenalectomy on CCD: evidence for differential response of two cell types.Am J Physiol Renal Fluid Electrolyte Physiol. 1987; 253: F742-F752PubMed Google Scholar, 23.Sansom S.C. O'Neil R.G. Effects of mineralocorticoids on transport properties of cortical collecting duct basolateral membrane.Am J Physiol Renal Fluid Electrolyte Physiol. 1986; 251: F743-F757PubMed Google Scholar We measured net fluxes of Na+, K+, and water in the CCD segments perfused in vitro using standard techniques previously described in detail.4.Muto S. Giebisch G. Sansom S. An acute increase of peritubular K stimulates K transport through cell pathways of CCT.Am J Physiol Renal Fluid Electrolyte Physiol. 1988; 255: F108-F114PubMed Google Scholar, 20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar Concentrations of Na+, K+, and inulin of the luminal perfusate and collected fluid were simultaneously measured using a continuous flow ultramicrofluorometer (Nanoflo; W-P Instruments Inc., Sarasota, FL, USA)20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar and an ultramicroflame photometer (AFA-707-D; APEL, Saitama, Japan),20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar respectively. Sodium green (Molecular probe, Eugene, OR, USA) and fluorescein isothiocyanate-inulin (Sigma, St Louis, MO, USA) were used as markers of Na+ and inulin, respectively. We calculated net water flux (Jv, nl/mm/min) using standard flux equations, as described previously.4.Muto S. Giebisch G. Sansom S. An acute increase of peritubular K stimulates K transport through cell pathways of CCT.Am J Physiol Renal Fluid Electrolyte Physiol. 1988; 255: F108-F114PubMed Google Scholar, 20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar Values of Jv greater than±0.1 nl/mm/min were assumed to represent mechanical leaks and were discarded. The rates of net cation transport (nl/mm/min) were calculated using standard flux equations, as previously described.4.Muto S. Giebisch G. Sansom S. An acute increase of peritubular K stimulates K transport through cell pathways of CCT.Am J Physiol Renal Fluid Electrolyte Physiol. 1988; 255: F108-F114PubMed Google Scholar, 20.Muto S. Tsuruoka S. Miyata Y. et al.Effect of trimethoprim-sulfamethoxazole on Na+ and K+ transport properties in the rabbit cortical collecting duct perfused in vitro.Nephron Physiol. 2006; 102: 51-60Crossref Scopus (9) Google Scholar The luminal flow rate was adjusted to approximately 7 nl/min by regulating the hydrostatic perfusion pressure. Each net flux was measured three times and averaged to yield a single measurement. The length of the tubule used for flux measurements was approximately 1.0 mm. Details on our techniques for measuring pHi in isolated perfused tubules have been published elsewhere.19.Muto S. Miyata Y. Imai M. et al.Troglitazone stimulates basolateral rheogenic Na+/HCO3- cotransport activity in rabbit proximal tubule S2 segments.Exp Nephrol. 2001; 9: 191-197Crossref PubMed Google Scholar The isolated perfused CCDs were exposed from the bath to the solution containing 2.5 mM K+ plus BCECF-AM (Dojindo, Kumamoto, Japan; 10 μM). After a 15-min dye-loading period at 37 °C, the dye was washed out. The pHi was then measured microfluorometrically by alternately exciting the dye with a 7.5-μm diameter spot of light at 440 and 490 nm while monitoring the emission at 530 nm.19.Muto S. Miyata Y. Imai M. et al.Troglitazone stimulates basolateral rheogenic Na+/HCO3- cotransport activity in rabbit proximal tubule S2 segments.Exp Nephrol. 2001; 9: 191-197Crossref PubMed Google Scholar The resulting fluorescence-excitation ratios were converted to pHi values as described,19.Muto S. Miyata Y. Imai M. et al.Troglitazone stimulates basolateral rheogenic Na+/HCO3- cotransport activity in rabbit proximal tubule S2 segments.Exp Nephrol. 2001; 9: 191-197Crossref PubMed Google Scholar using the high-K+/nigericin technique.25.Thomas J.A. Buchsbaum R.N. Zimniak A. et al.Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.Biochemistry. 1979; 18: 2210-2218Crossref PubMed Scopus (1730) Google Scholar In this study, the tubular lumen was also perfused at flow rates of approximately 7 nl/min by regulating the hydrostatic perfusion pressure. Data are shown as means±s.e.m., and paired or nonpaired Student's t-test was used to determine the significance between the two groups. Statistical significance was taken as P<0.05. All the authors declared no competing interest. This work was supported by a grant from the Salt Science Foundation (S Muto), and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (S Muto) and by National Institutes of Health Grant DK 47402 (WH Wang).
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