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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
2006; Elsevier BV; Volume: 70; Issue: 1 Linguagem: Inglês
10.1038/sj.ki.5000388
ISSN1523-1755
AutoresMatthew A. Bailey, Alessandra Cantone, Q. Yan, Gordon G. MacGregor, Qiang Leng, José Benedito Oliveira Amorim, T. Wang, Steven Hébert, Gerhard Giebisch, Gerhard Malnic,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoType II Bartter's syndrome is a hereditary hypokalemic renal salt-wasting disorder caused by mutations in the ROMK channel (Kir1.1; Kcnj1), mediating potassium recycling in the thick ascending limb of Henle's loop (TAL) and potassium secretion in the distal tubule and cortical collecting duct (CCT). Newborns with Type II Bartter are transiently hyperkalemic, consistent with loss of ROMK channel function in potassium secretion in distal convoluted tubule and CCT. Yet, these infants rapidly develop persistent hypokalemia owing to increased renal potassium excretion mediated by unknown mechanisms. Here, we used free-flow micropuncture and stationary microperfusion of the late distal tubule to explore the mechanism of renal potassium wasting in the Romk-deficient, Type II Bartter's mouse. We show that potassium absorption in the loop of Henle is reduced in Romk-deficient mice and can account for a significant fraction of renal potassium loss. In addition, we show that iberiotoxin (IBTX)-sensitive, flow-stimulated maxi-K channels account for sustained potassium secretion in the late distal tubule, despite loss of ROMK function. IBTX-sensitive potassium secretion is also increased in high-potassium-adapted wild-type mice. Thus, renal potassium wasting in Type II Bartter is due to both reduced reabsorption in the TAL and K secretion by max-K channels in the late distal tubule. Type II Bartter's syndrome is a hereditary hypokalemic renal salt-wasting disorder caused by mutations in the ROMK channel (Kir1.1; Kcnj1), mediating potassium recycling in the thick ascending limb of Henle's loop (TAL) and potassium secretion in the distal tubule and cortical collecting duct (CCT). Newborns with Type II Bartter are transiently hyperkalemic, consistent with loss of ROMK channel function in potassium secretion in distal convoluted tubule and CCT. Yet, these infants rapidly develop persistent hypokalemia owing to increased renal potassium excretion mediated by unknown mechanisms. Here, we used free-flow micropuncture and stationary microperfusion of the late distal tubule to explore the mechanism of renal potassium wasting in the Romk-deficient, Type II Bartter's mouse. We show that potassium absorption in the loop of Henle is reduced in Romk-deficient mice and can account for a significant fraction of renal potassium loss. In addition, we show that iberiotoxin (IBTX)-sensitive, flow-stimulated maxi-K channels account for sustained potassium secretion in the late distal tubule, despite loss of ROMK function. IBTX-sensitive potassium secretion is also increased in high-potassium-adapted wild-type mice. Thus, renal potassium wasting in Type II Bartter is due to both reduced reabsorption in the TAL and K secretion by max-K channels in the late distal tubule. Neonatal or antenatal Bartter's syndrome represents a group of hereditary hypokalemic renal salt-wasting disorders caused by loss-of-function mutations in proteins that either mediate or modulate electrolyte transport in the thick ascending limb (TAL) of Henle's loop.1.Hebert S.C. Bartter syndrome.Curr Opin Nephrol Hypertens. 2003; 12: 527-532Crossref PubMed Scopus (210) Google Scholar, 2.Reinalter S.C. Jeck N. Peters M. Seyberth H.W. Pharmacotyping of hypokalaemic salt-losing tubular disorders.Acta Physiol Scand. 2004; 181: 513-521Crossref PubMed Scopus (40) Google Scholar Mutations in the low-conductance K channel, ROMK (Kir1.1; Kcnj1), cause Type II Bartter's syndrome,3.Karolyil L. Konrad M. Kockerling A. et al.Mutations in the gene encoding the inwardly-rectifying renal potassium channel, ROMK, cause the antenatal variant of Bartter syndrome: evidence for genetic heterogeneity.Hum Mol Genet. 1997; 6: 17-26Crossref PubMed Scopus (177) Google Scholar, 4.Simon D.B. Karet F.E. Rodriguez-Soriano J. et al.Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK.Nat Genet. 1996; 14: 152-156Crossref PubMed Scopus (677) Google Scholar characterized by natriuresis, diuresis, hypokalemic metabolic alkalosis, elevated prostaglandin E2 and renin, and an elevated Ca2+ excretion leading to nephrocalcinosis. ROMK channels play critical roles in salt transport processes in the TAL. About 25% of Na and K that are ultrafiltered at the glomerulus are reabsorbed in the loop of Henle5.Malnic G. Klose R.M. Giebisch G. Microperfusion study of distal tubular potassium and sodium transfer in rat kidney.Am J Physiol. 1966; 211: 548-559PubMed Google Scholar and ROMK provides the pathway for apical K recycling, which is necessary for supplying luminal K to the Na–K–2Cl cotransporter, NKCC2.6.Hebert S.C. Mount D.B. Gamba G. Molecular physiology of cation-coupled Cl− cotransport: the SLC12 family.Pflugers Arch. 2003; 447: 594-602PubMed Google Scholar In addition, this apical K current generates a lumen-positive potential which drives the paracellular reabsorption of a significant fraction of Na and K7.Greger R. Schlatter E. Presence of luminal K+, a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle's loop of rabbit kidney.Pflugers Arch. 1981; 392: 92-94Crossref PubMed Scopus (180) Google Scholar, 8.Hebert S.C. Andreoli T.E. Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl− absorption.J Genet Physiol. 1986; 87: 567-590Crossref PubMed Scopus (48) Google Scholar and is critical for transcellular NaCl reabsorption by furnishing the apical part of a transcellular current pathway required for basolateral Cl exit.8.Hebert S.C. Andreoli T.E. Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl− absorption.J Genet Physiol. 1986; 87: 567-590Crossref PubMed Scopus (48) Google Scholar Thus, K channels play a crucial role in the reabsorption of a major fraction of NaCl and K in the TAL, and their absence results in a loop diuretic-like effect, accounting for the natriuresis of Type II Bartter. Patch-clamp studies in the Type II Bartter's mouse (Romk−/−) with loss-of-function mutation in ROMK9.Lorenz J.N. Baird N.R. Judd L.M. et al.Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter's syndrome.J Biol Chem. 2002; 277: 37871-37880Crossref PubMed Scopus (146) Google Scholar have demonstrated the absence of K channels in the apical membrane of the TAL cells.10.Lu M. Wang T. Yan Q. et al.ROMK is required for expression of the 70pS K channel in the thick ascending limb.Am J Physiol Renal Physiol. 2004; 286: F490-F495Crossref PubMed Scopus (42) Google Scholar, 11.Lu M. Wang T. Yan Q. et al.Absence of small-conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice.J Biol Chem. 2002; 277: 37881-37887Crossref PubMed Scopus (145) Google Scholar Micropuncture studies of early distal tubules in Romk−/− mice show increased Cl and fluid delivery into the early distal tubule,9.Lorenz J.N. Baird N.R. Judd L.M. et al.Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter's syndrome.J Biol Chem. 2002; 277: 37871-37880Crossref PubMed Scopus (146) Google Scholar consistent with diminished salt reabsorption along Henle's loop. These findings support the view that loss of ROMK function contributes to diminished tubule NaCl reabsorption in the loop of Henle and ultimately to natriuresis. ROMK channels also provide the major K-secretory pathway for regulated K excretion by the connecting segment (CNT), and initial collecting and cortical collecting ducts (CCTs12.Frindt G. Palmer L.G. Ca-activated K channels in apical membrane of mammalian CCT, and their role in K secretion.Am J Physiol (Renal Fluid Electrolyte Physiol). 1987; 252-21: F458-F467Google Scholar, 13.Frindt G. Palmer L.G. Apical potassium channels in the rat connecting tubule.Am J Physiol Renal Physiol. 2004; 287: F1030-F1037Crossref PubMed Scopus (67) Google Scholar, 14.Gray D.A. Frindt G. Palmer L.G. Quantification of K+ secretion through apical low-conductance K channels in the CCD.Am J Physiol Renal Physiol. 2005; 289: F117-F126Crossref PubMed Scopus (36) Google Scholar, 15.Hebert S.C. Desir G. Giebisch G. Wang W. Molecular diversity and regulation of renal potassium channels.Physiol Rev. 2005; 85: 319-371Crossref PubMed Scopus (249) Google Scholar), and this channel activity is lost from principal cells in the CCT in ROMK-deficient mice.11.Lu M. Wang T. Yan Q. et al.Absence of small-conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice.J Biol Chem. 2002; 277: 37881-37887Crossref PubMed Scopus (145) Google Scholar In the CCT, the surface expression of ROMK channels, and thereby the apical K conductance of principal cells, is regulated by dietary K intake.16.Lin D.H. Sterling H. Wang W.H. The protein tyrosine kinase-dependent pathway mediates the effect of K intake on renal K secretion.Physiology (Bethesda). 2005; 20: 140-146Crossref PubMed Scopus (25) Google Scholar A high dietary K intake increases the number of active ROMK channels in apical membranes of principal cells, and thus enhances the K secretory capacity of the CCT. In contrast, ROMK activity does not appear to be increased by a high K intake in the CNT.13.Frindt G. Palmer L.G. Apical potassium channels in the rat connecting tubule.Am J Physiol Renal Physiol. 2004; 287: F1030-F1037Crossref PubMed Scopus (67) Google Scholar Infants with the Type II Bartter's genotype often exhibit a transient hyperkalemic phenotype,17.Cho J.T. Guay-Woodford L.M. Heterozygous mutations of the gene for Kir 1.1 (ROMK) in antenatal Bartter syndrome presenting with transient hyperkalemia, evolving to a benign course.J Korean Med Sci. 2003; 18: 65-68Crossref PubMed Scopus (9) Google Scholar, 18.Finer G. Shalev H. Birk O.S. et al.Transient neonatal hyperkalemia in the antenatal (ROMK defective) Bartter syndrome.J Pediatr. 2003; 142: 318-323Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar consistent with the loss of ROMK channels in the distal tubule and collecting duct. Yet, neither older Type II Bartter's infants19.Peters M. Jeck N. Reinalter S. et al.Clinical presentation of genetically defined patients with hypokalemic salt-losing tubulopathies.Am J Med. 2002; 112: 183-190Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 2.Reinalter S.C. Jeck N. Peters M. Seyberth H.W. Pharmacotyping of hypokalaemic salt-losing tubular disorders.Acta Physiol Scand. 2004; 181: 513-521Crossref PubMed Scopus (40) Google Scholar nor Romk−/− mice9.Lorenz J.N. Baird N.R. Judd L.M. et al.Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter's syndrome.J Biol Chem. 2002; 277: 37871-37880Crossref PubMed Scopus (146) Google Scholar, 11.Lu M. Wang T. Yan Q. et al.Absence of small-conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice.J Biol Chem. 2002; 277: 37881-37887Crossref PubMed Scopus (145) Google Scholar are hyperkalemic, but rather are kaluretic and hypokalemic. The mechanism for development of this kaluresis is poorly understood in the antenatal Type II Bartter's genotype. However, a high-conductance K channel has also been identified in principal20.Hunter M. Lopes A.G. Boulpaep E.L. Giebisch G. Single channel recordings of calcium-activated potassium channels in the apical emmbrane of rabbit cortical collecting tubules.Proc Natl Acad Sci USA. 1984; 81: 4237-4239Crossref PubMed Scopus (128) Google Scholar and intercalated cells21.Pacha J. Frindt G. Sackin H. Palmer L.G. Apical maxi K channels in intercalated cells of CCT.Am J Physiol. 1991; 261: F696-F705PubMed Google Scholar in the CCT and in the late distal tubule (LDT; including the connecting segment13.Frindt G. Palmer L.G. Apical potassium channels in the rat connecting tubule.Am J Physiol Renal Physiol. 2004; 287: F1030-F1037Crossref PubMed Scopus (67) Google Scholar). This maxi-K channel is sensitive to iberiotoxin (IBTX) and has been suggested to participate in distal K secretion during high tubule fluid flow conditions22.Liu W. Xu S. Woda C. et al.Effect of flow and stretch on the [Ca2+]i response of principal and intercalated cells in cortical collecting duct.Am J Physiol Renal Physiol. 2003; 285: F998-F1012Crossref PubMed Scopus (187) Google Scholar, 23.Satlin L.M. Developmental regulation of expression of renal potassium secretory channels.Curr Opin Nephrol Hypertens. 2004; 13: 445-450Crossref PubMed Scopus (35) Google Scholar, 24.Woda C.B. Bragin A. Kleyman T.R. Satlin L.M. 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 and during elevations in lumen arginine vasopressin via apical V1 receptors.25.Amorim J.B. Malnic G. V1 receptors in luminal action of vasopressin on distal K+ secretion.Am J Physiol Renal Physiol. 2000; 278: F809-F816PubMed Google Scholar, 26.Amorim J.B. Musa-Aziz R. Mello-Aires M. Malnic G. Signaling path of the action of AVP on distal K+ secretion.Kidney Int. 2004; 66: 696-704Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Although a high distal flow is likely present in the diuretic Bartter's infants and mice and this could activate maxi-K channels, their role in K secretion is unclear as the ROMK channel appears to be sufficient to account for K secretion under most conditions.12.Frindt G. Palmer L.G. Ca-activated K channels in apical membrane of mammalian CCT, and their role in K secretion.Am J Physiol (Renal Fluid Electrolyte Physiol). 1987; 252-21: F458-F467Google Scholar, 14.Gray D.A. Frindt G. Palmer L.G. Quantification of K+ secretion through apical low-conductance K channels in the CCD.Am J Physiol Renal Physiol. 2005; 289: F117-F126Crossref PubMed Scopus (36) Google Scholar Thus, the role of maxi-K channels in the middle-to-late distal tubule as a mechanism for K secretion in Type II Bartter is unclear and has not been explored. To assess the mechanisms for renal K loss in the Type II Bartter's mouse, we used both micropuncture and stationary microperfusion techniques to assess K transport in the loop of Henle and to quantify K secretion in the LDT. We observed that the high renal K excretion is maintained in Romk−/− mice both by a decreased reabsorption of K in the loop of Henle and by the activation of maxi-K channels in the LDT. In addition, we found that IBTX-sensitive maxi-K channels also contribute significantly to K secretion in high-potassium-diet-adapted wild-type mice. The latter suggests that the IBTX-sensitive maxi-K channel activity can be upregulated by a high dietary K intake in the CCD in normal mice. Both the proximal tubule and the loop of Henle, including the TAL, mediate the bulk of potassium reabsorption. Free-flow micropuncture studies show that a large fraction of filtered potassium is reabsorbed before the superficial distal tubules.27.Malnic G. Klose R. Giebisch G. Micropuncture study of renal potassium excretion in the rat.Am J Physiol. 1964; 206: 674-686PubMed Google Scholar Normally, the potassium concentration in early distal tubule fluid is significantly reduced below plasma levels. Here, we compare K activity in the early distal tubules of wild-type and Romk−/− mice as a reflection of K transport in upstream tubule segments. The low values of tubule negativity confirms the early distal tubule recording site (Figure 1a).28.Costanzo L.S. Windhager E.E. Calcium and sodium transport by the distal convoluted tubule of the rat.Am J Physiol (Renal Fluid Electrolyte Physiol). 1978; 235: F492-F506PubMed Google Scholar, 29.Wright F.S. Increasing magnitude of electrical potential along the renal distal tubule.Am J Physiol. 1971; 220: 624-638PubMed Google Scholar The lumen potassium activity was 2.7±0.5 mM (n=7) in Romk+/+ animals (Figure 1b), comparable to the value in Sprague–Dawley rats under similar experimental conditions.27.Malnic G. Klose R. Giebisch G. Micropuncture study of renal potassium excretion in the rat.Am J Physiol. 1964; 206: 674-686PubMed Google Scholar Potassium activity was significantly elevated to 8.6±1.6 mM (n=7; P<0.01) in Romk−/− animals. Considering that the delivery of tubule fluid to the early distal tubule is also significantly higher in Romk−/− mice,9.Lorenz J.N. Baird N.R. Judd L.M. et al.Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter's syndrome.J Biol Chem. 2002; 277: 37871-37880Crossref PubMed Scopus (146) Google Scholar these results show that potassium reabsorption, most likely in the TAL, is sharply reduced. The LDT plays a prominent and critical role in maintaining Na and K homeostasis.30.Meneton P. Loffing J. Warnock D.G. Sodium and potassium handling by the aldosterone-sensitive distal nephron: the pivotal role of the distal and connecting tubule.Am J Physiol Renal Physiol. 2004; 287: F593-F601Crossref PubMed Scopus (142) Google Scholar, 31.Rubera I. Loffing J. Palmer L.G. et al.Collecting duct-specific gene inactivation of αENaC in the mouse kidney does not impair sodium and potassium balance.J Clin Invest. 2003; 112: 554-565Crossref PubMed Scopus (170) Google Scholar Thus, we used stationary microperfusion to assess whether K secretion into the LDT contributes to the kaluresis observed in Romk−/− mice. In previous stationary microperfusion studies in rats, approximately 80% of K secretion was Ba2+-sensitive,32.Amorim J.B. Bailey M.A. Musa-Aziz R. et al.Role of luminal anion and pH in distal tubule potassium secretion.Am J Physiol Renal Physiol. 2003; 284: F381-F388Crossref PubMed Scopus (42) Google Scholar, 25.Amorim J.B. Malnic G. V1 receptors in luminal action of vasopressin on distal K+ secretion.Am J Physiol Renal Physiol. 2000; 278: F809-F816PubMed Google Scholar and therefore mediated by potassium channels. Although K–Cl cotransport can contribute to K secretion into the rat LDT under low luminal Cl conditions (<20 mM Cl), the contribution of this furosemide- and DIOA ((dihydroindenyl)oxy alkanoic acid)-sensitive pathway to K secretion is negligible with the 100 mM luminal Cl solutions used in the present studies.32.Amorim J.B. Bailey M.A. Musa-Aziz R. et al.Role of luminal anion and pH in distal tubule potassium secretion.Am J Physiol Renal Physiol. 2003; 284: F381-F388Crossref PubMed Scopus (42) Google Scholar A similar fraction of Ba2+-sensitive K secretion (80%) was observed in isolated perfused CCT segments from rat33.Schafer J.A. Troutman S.L. Potassium transport in cortical collecting tubules from mineralocorticoid-treated rat.Am J Physiol (Renal Fluid Electrolyte Physiol). 1987; 253: F76-F88PubMed Google Scholar or rabbit.34.Muto S. Giebisch G. Sansom S. An acute increase of peritubular K stimulates K transport through cell pathways of CCT.Am J Physiol. 1988; 255: F108-F114PubMed Google Scholar The remaining 20% of measured K secretion in our in vivo studies is thought to be passive and mediated by the large lumen-negative potential due to amiloride-sensitive Na transport. At least two types of K channels contribute to Ba2+-sensitive K secretion into the LDT: ROMK and maxi-K channels.32.Amorim J.B. Bailey M.A. Musa-Aziz R. et al.Role of luminal anion and pH in distal tubule potassium secretion.Am J Physiol Renal Physiol. 2003; 284: F381-F388Crossref PubMed Scopus (42) Google Scholar, 25.Amorim J.B. Malnic G. V1 receptors in luminal action of vasopressin on distal K+ secretion.Am J Physiol Renal Physiol. 2000; 278: F809-F816PubMed Google Scholar, 26.Amorim J.B. Musa-Aziz R. Mello-Aires M. Malnic G. Signaling path of the action of AVP on distal K+ secretion.Kidney Int. 2004; 66: 696-704Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 13.Frindt G. Palmer L.G. Apical potassium channels in the rat connecting tubule.Am J Physiol Renal Physiol. 2004; 287: F1030-F1037Crossref PubMed Scopus (67) Google Scholar We used IBTX, a specific inhibitor of maxi-K channels,35.Candia S. Garcia M.L. Latorre R. Mode of action of iberiotoxin, a potent blocker of the large conductance Ca2+-activated K+ channel.Biophys J. 1992; 63: 583-590Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 36.Galvez A. Gimenez-Gallego G. Reuben J.P. et al.Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus.J Biol Chem. 1990; 265: 11083-11090Abstract Full Text PDF PubMed Google Scholar to differentiate between these mechanisms of K secretion in the LDT. To establish the lack of sensitivity of ROMK to IBTX, we investigated the effect of this inhibitor on ROMK expressed in Xenopus laevis oocytes. IBTX up to 1 μM had no effect on whole-cell currents in X. laevis oocytes expressing ROMK2 (Figure 2). Similar results were obtained with ROMK1 (data not shown). Figure 3 shows representative K activity ([K]) traces illustrating the rise in lumen K to steady-state values in the absence or presence of IBTX in Romk+/+ (Figure 3a and b) and Romk−/− (Figure 3c) mice. The rate of rise in [K] was slower and the steady-state [K] values lower in Romk−/− compared to Romk+/+ mice. IBTX slowed the rate of rise in [K] in both Romk genotypes. However, in Romk+/+ mice, the response to IBTX was variable such that a reduced rate of rise in [K] was only observed in about half of the mice (Figure 3a and b). This may reflect that we had not achieved complete genetic homogeneity in our colony from the mixed genetic background of the original mice (129/SvJ and C57/Bl-6). Technical issues are unlikely to account for the variability seen in Romk+/+ mice as consistent IBTX insensitivity was observed in C57/Bl-6 mice on the same 1.2% K diet (JK in Table 2).Table 2Effect of a high-K (10%) diet on the IBTX-sensitive component of potassium secretionC57/Bl-6C57/Bl-6Romk+/+K diet1.2%10%10%JK (nmol cm−2 s−1) Control-0.63±0.06 (11)-0.86±0.04# (14)-0.91±0.16 (6) IBTX-0.61±0.07 (11)-0.57±0.07** (14)-0.47±0.11* (6)[K+] (mM) Control17.7±2.0 (11)20.9±1.2 (14)20.7±1.8 (6) IBTX16.4±1.7 (11)20.7±1.2 (14)17.5±1.3 (6)T1/2 (s) Control9.2±1.9 (11)6.7±0.5# (14)9.5±1.1 (6) IBTX8.3±1.7 (11)12.3±1.5** (14)34.8±10.1* (6)PD (mV) Control-40.6±4.3 (7)-39.5±3.8 (6)-27.4±3.4 (3)IBTX, iberiotoxin; PD, potential difference.Data are means±s.e.m. JK, t1/2, and [K+]s are as defined in equation (1).*P<0.01 vs control; **P<0.01 vs control; #P<0.001 vs normal diet for the genotype.Numbers in parentheses indicate numbers of tubules. Open table in a new tab IBTX, iberiotoxin; PD, potential difference. Data are means±s.e.m. JK, t1/2, and [K+]s are as defined in equation (1). *P<0.01 vs control; **P<0.01 vs control; #P<0.001 vs normal diet for the genotype. Numbers in parentheses indicate numbers of tubules. Table 1 and Figure 4a summarize the data for JK, t1/2, maximal [K], and potential difference in Romk genotypes on the 1.2% diet. In Romk+/+ mice, JK averaged -0.65 nmol cm−2 s−1, a value similar to that in rats under comparable experimental conditions32.Amorim J.B. Bailey M.A. Musa-Aziz R. et al.Role of luminal anion and pH in distal tubule potassium secretion.Am J Physiol Renal Physiol. 2003; 284: F381-F388Crossref PubMed Scopus (42) Google Scholar and to JK in C57/Bl-6 (Table 2). In these mice, JK was reduced by ∼50% with IBTX. In Romk−/− mice, JK was significantly lower (-0.28 nmol cm−2 s−1) than in wild-type littermates, and this secretory K flux was abolished by IBTX. The absence of K secretion in Romk−/− mice exposed to IBTX indicates that passive back flux of K from blood to lumen must be quite small in these mice, despite the significant lumen-negative driving force (-40.2 mV; Table 1). Thus, the LDT of ROMK-deficient mice continues to secrete K, albeit at a significantly reduced rate, by an IBTX-sensitive mechanism consistent with maxi-K channels.Table 1Contribution of IBTX-sensitive and IBTX-insensitive pathways to potassium secretion in wild-type (Romk+/+) and ROMK-deficient (Romk−/−) miceRomk+/+Romk−/−JK (nmol cm−2 s−1) Control-0.65±0.14 (12)-0.28±0.11# (7) IBTX-0.35±0.15* (11)-0.02±0.01* (7)[K+] (mM) Control20.9±2.1 (12)14.6±2.9# (7) IBTX11.7±2.0* (11)6.2±1.6*,# (7)t1/2 (s) Control14.3±2.6 (12)22.6±5.7 (7) IBTX31.3±10.1* (11)115.9±45.6*,# (7)PD (mV) Control-37.7±5.0 (10)-40.2±4.9 (9)IBTX, iberiotoxin.Data are means±s.e.m. JK, t1/2, and [K+]s are as defined in equation (1).*P<0.05 IBTX vs control; #P<0.05 Romk+/+ vs Romk−/−.Numbers in parentheses indicate numbers of tubules. PD is the free-flow potential difference. Open table in a new tab IBTX, iberiotoxin. Data are means±s.e.m. JK, t1/2, and [K+]s are as defined in equation (1). *P<0.05 IBTX vs control; #P<0.05 Romk+/+ vs Romk−/−. Numbers in parentheses indicate numbers of tubules. PD is the free-flow potential difference. In the CCT, but not CNT, ROMK channel activity increases several fold in rats fed a high-K (∼10%) diet,15.Hebert S.C. Desir G. Giebisch G. Wang W. Molecular diversity and regulation of renal potassium channels.Physiol Rev. 2005; 85: 319-371Crossref PubMed Scopus (249) Google Scholar and can quantitatively account for the increased rate of K secretion in this nephron segment.14.Gray D.A. Frindt G. Palmer L.G. Quantification of K+ secretion through apical low-conductance K channels in the CCD.Am J Physiol Renal Physiol. 2005; 289: F117-F126Crossref PubMed Scopus (36) Google Scholar However, a recent study suggested that dietary K regulates expression of maxi-K channels in the rabbit CCT,37.Najjar F. Zhou H. Morimoto T. et al.Dietary K+ regulates apical membrane expression of maxi-K channels in rabbit cortical collecting duct.Am J Physiol Renal Physiol. 2005; 289: F922-F932Crossref PubMed Scopus (95) Google Scholar and therefore an IBTX-sensitive flux could contribute to K secretion in the CCT or LDT in high-K-adapted animals. Thus, we examined if JK increases in the LDT of Romk+/+ mice adapted to a 10% K diet and if the IBTX-sensitive component of JK contributes to the increase in K secretion. We also examined the role of max-K channels in C57/Bl-6 mice, which contributes to the mixed genetic background of our inbred Romk−/− mice. In both Romk+/+ and C57/Bl-6 mice, JK significantly increased to a similar magnitude on the high-K (10%) diet (Tables 1 and 2; Figure 4). In both species, this increase was largely (Romk+/+) or completely (C57/Bl-6) accounted for by an increase in the IBTX-sensitive flux. This supports an important role for maxi-K channels in the high-K adaptive response of the LDT. Individuals with Type II Bartter's syndrome due to mutations in the ROMK gene (Kcnj1) are often hyperkalemic during the first few weeks following birth, but rapidly develop a sustained hypokalemia owing to renal potassium wasting.17.Cho J.T. Guay-Woodford L.M. Heterozygous mutations of the gene for Kir 1.1 (ROMK) in antenatal Bartter syndrome presenting with transient hyperkalemia, evolving to a benign course.J Korean Med Sci. 2003; 18: 65-68Crossref PubMed Scopus (9) Google Scholar, 18.Finer G. Shalev H. Birk O.S. et al.Transient neonatal hyperkalemia in the antenatal (ROMK defective) Bartter syndrome.J Pediatr. 2003; 142: 318-323Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 38.Jeck N. Derst C. Wischmeyer E. et al.Functional heterogeneity of ROMK mutations linked to hyperprostaglandin E syndrome.Kidney Int. 2001; 59: 1803-1811Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 19.Peters M. Jeck N. Reinalter S. et al.Clinical presentation of genetically defined patients with hypokalemic salt-losing tubulopathies.Am J Med. 2002; 112: 183-190Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar Loss of function of the ROMK K secretory channel can account for the initial hyperkalemia, but the mechanism(s) contributing to the subsequent kaluresis has remained unclear. The present studies in the ROMK-deficient mouse model of Type II Bartter's syndrome demonstrate that the kaluresis which occurs in the absence of this 35 pS K secretory channel results from two processes (Figure 5): reduced K reabsorption in the TAL and sustained K secretion in the LDT via IBTX-sensitive maxi-K channels. Our studies also demonstrate that this IBTX-sensitive K flux provides a major component of increased K secretion in the LDT in response to a high-K diet. Thus, although ROMK potassium channels are crucial for K handling by the kidney, maxi-K channels expressed in the LDT contribute importantly to renal K excretion in response to elevations in potassium intake and to the increased K excretion in Type II Bartter's syndrome. The TAL normally reabsorbs approximately 20–25% of the filtered load of potassium by cotransport with Na and Cl via the Na–K–2Cl cotransporter, NKCC2.39.Hropot M. Fowler N. Karlmark B. Giebisch G. Tubular action of diuretics: distal effects on electrolyte transport and acidification.Kidney Int. 1985; 28: 477-489Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 40.Morgan T. Tadokoro M. Martin D. Berliner R.W. Effect of furosemide on Na+ and K+ transport studied by microperfusion of the rat nephron.Am J Physiol. 1970; 218: 292-297PubMed Google Scholar Interestingly, about 30% of normal NaCl reabsorption remains in ROMK-deficient mice,9.Lorenz J.N. Baird N.R. Judd L.M. et al.Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter's syndrome.J Biol Chem. 2002; 277: 37871-37880Crossref PubMed Scopus (146) Google Scholar despite loss of all apical K conductance10.Lu M. Wang T. Yan Q. et al.ROMK is required for expression of the 70pS K c
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