Defective ENaC Processing and Function in Tissue Kallikrein-deficient Mice
2007; Elsevier BV; Volume: 283; Issue: 8 Linguagem: Inglês
10.1074/jbc.m705664200
ISSN1083-351X
AutoresNicolas Picard, Dominique Eladari, Soumaya El Moghrabi, Carole Planès, Soline Bourgeois, Pascal Houillier, Qing Wang, Michel Burnier, Georges Deschênes, Mark A. Knepper, Pierre Meneton, Régine Chambrey,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoAn inverse relationship exists between urinary tissue kallikrein (TK) excretion and blood pressure in humans and rodents. In the kidney TK is synthesized in large amounts in the connecting tubule and is mainly released into the urinary fluid where its function remains unknown. In the present study mice with no functional gene coding for TK (TK–/–) were used to test whether the enzyme regulates apically expressed sodium transporters. Semiquantitative immunoblotting of the renal cortex revealed an absence of the 70-kDa form of γ-ENaC in TK–/– mice. Urinary Na+ excretion after amiloride injection was blunted in TK–/– mice, consistent with reduced renal ENaC activity. Amiloride-sensitive transepithelial potential difference in the colon, where TK is also expressed, was decreased in TK–/– mice, whereas amiloride-sensitive alveolar fluid clearance in the lung, where TK is not expressed, was unchanged. In mice lacking the B2 receptor for kinins, the abundance of the 70-kDa form of γ-ENaC was increased, indicating that its absence in TK–/– mice is not kinin-mediated. Incubation of membrane proteins from renal cortex of TK–/– mice with TK resulted in the appearance of the 70-kDa band of the γ-ENaC, indicating that TK was able to promote γ-ENaC cleavage in vitro. Finally, in mouse cortical collecting ducts isolated and microperfused in vitro, the addition of TK in the luminal fluid increased significantly intracellular Na+ concentration, consistent with an activation of the luminal entry of the cation. The results demonstrate that TK, like several other proteases, can activate ENaC in the kidney and the colon. An inverse relationship exists between urinary tissue kallikrein (TK) excretion and blood pressure in humans and rodents. In the kidney TK is synthesized in large amounts in the connecting tubule and is mainly released into the urinary fluid where its function remains unknown. In the present study mice with no functional gene coding for TK (TK–/–) were used to test whether the enzyme regulates apically expressed sodium transporters. Semiquantitative immunoblotting of the renal cortex revealed an absence of the 70-kDa form of γ-ENaC in TK–/– mice. Urinary Na+ excretion after amiloride injection was blunted in TK–/– mice, consistent with reduced renal ENaC activity. Amiloride-sensitive transepithelial potential difference in the colon, where TK is also expressed, was decreased in TK–/– mice, whereas amiloride-sensitive alveolar fluid clearance in the lung, where TK is not expressed, was unchanged. In mice lacking the B2 receptor for kinins, the abundance of the 70-kDa form of γ-ENaC was increased, indicating that its absence in TK–/– mice is not kinin-mediated. Incubation of membrane proteins from renal cortex of TK–/– mice with TK resulted in the appearance of the 70-kDa band of the γ-ENaC, indicating that TK was able to promote γ-ENaC cleavage in vitro. Finally, in mouse cortical collecting ducts isolated and microperfused in vitro, the addition of TK in the luminal fluid increased significantly intracellular Na+ concentration, consistent with an activation of the luminal entry of the cation. The results demonstrate that TK, like several other proteases, can activate ENaC in the kidney and the colon. Tissue kallikrein 5In this paper, tissue kallikrein indicates the product of the mouse klk1 gene (accession number NM_010639); synonyms are KLK1, Kal, mGk-6, renal kallikrein, and Klk1b6. (TK) 6The abbreviations used are: TKtissue kallikreinPDpotential differenceSBFIsodium-binding benzofuran isophthalateCCDcortical collecting ductASDNaldosterone-sensitive distal nephronWTwild typeAFCalveolar fluid clearanceCAPchannel-activating protease. is a serine protease that generates kinins locally in many organs, including the kidney, colon, and arteries. In the kidney, TK that is synthesized in large amounts by connecting tubule cells (1Proud D. Knepper M.A. Pisano J.J. Am. J. Physiol. 1983; 244: F510-F515PubMed Google Scholar) is mainly secreted into the urinary fluid and to a lesser extent to the peritubular interstitium. In the renal interstitium it cleaves locally produced kininogen to yield bradykinin that in turn can activate type-2 (B2) bradykinin receptors. Bradykinin-dependent activation of B2 receptor increases sodium excretion by inhibiting sodium reabsorption in the collecting duct (2Tomita K. Pisano J.J. Knepper M.A. J. Clin. Investig. 1985; 76: 132-136Crossref PubMed Scopus (255) Google Scholar). Therefore, the renal kallikrein-kinin system is expected to play a role in renal NaCl balance and blood pressure regulation. Patients with essential hypertension have lower kallikrein levels in their urine (3Lechi A. Covi G. Lechi C. Corgnati A. Arosio E. Zatti M. Scuro L.A. Clin. Sci. Mol. Med. 1978; 55: 51-55PubMed Google Scholar, 4Seino M. Abe K. Otsuka Y. Saito T. Irokawa N. Tohoku J. Exp. Med. 1975; 116: 359-367Crossref PubMed Scopus (51) Google Scholar), and mutant mice lacking B2 receptor also exhibit salt-sensitive hypertension (5Alfie M.E. Sigmon D.H. Pomposiello S.I. Carretero O.A. Hypertension. 1997; 29: 483-487Crossref PubMed Google Scholar). However, inactivation of the TK gene in the mouse does not alter blood pressure (6Meneton P. Bloch-Faure M. Hagege A.A. Ruetten H. Huang W. Bergaya S. Ceiler D. Gehring D. Martins I. Salmon G. Boulanger C.M. Nussberger J. Crozatier B. Gasc J.M. Heudes D. Bruneval P. Doetschman T. Menard J. Alhenc-Gelas F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2634-2639Crossref PubMed Scopus (141) Google Scholar) even though the decrease in renal and urinary kallikrein activity in TK-deficient mice reproduces the phenotype that has been repeatedly associated with hypertension in human and rat studies (7Berry T.D. Hasstedt S.J. Hunt S.C. Wu L.L. Smith J.B. Ash K.O. Kuida H. Williams R.R. Hypertension. 1989; 13: 3-8Crossref PubMed Scopus (168) Google Scholar, 8Margolius H.S. Geller R. De Jong W. Pisano J.J. Sjoerdsma A. Circ. Res. 1972; 30: 358-362Crossref PubMed Scopus (123) Google Scholar, 9Margolius H.S. Geller R. Pisano J.J. Sjoerdsma A. Lancet. 1971; 2: 1063-1065Abstract PubMed Scopus (262) Google Scholar). This finding suggests that low urinary kallikrein excretion observed in hypertensive patients is not a primary cause of high blood pressure (HBP) but rather a consequence of hypertension or of HBP-associated renal defects. An alternative explanation is that TK-deficient mice develop compensatory mechanisms to keep blood pressure at normal levels. tissue kallikrein potential difference sodium-binding benzofuran isophthalate cortical collecting duct aldosterone-sensitive distal nephron wild type alveolar fluid clearance channel-activating protease. The role of the large amount of TK that is secreted into the urinary fluid remains unknown. One possibility would be that this protease acts directly on the different transporters expressed at the apical side of the tubular cells to modulate their activities. Apical sodium reabsorption along the renal tubule is achieved through multiple sodium transporters. Particularly, in the aldosterone-sensitive distal nephron (ASDN, i.e. distal convoluted tubule (DCT2), connecting tubule, and collecting duct), the amiloride-sensitive epithelial Na+ channel ENaC, consisting of α-, β-, and γ-subunits, mediates Na+ uptake across the apical plasma membrane of principal cells and connecting tubule cells (10Loffing J. Kaissling B. Am. J. Physiol. Renal Physiol. 2003; 284: 628-643Crossref PubMed Scopus (153) Google Scholar). In all renal tubule cells, the Na+ is extruded on the basolateral side in exchange for K+ by the Na+,K+-ATPase. Although sodium transport occurs throughout the length of the renal tubule, the fine regulation of sodium excretion occurs in the ASDN, mostly through aldosterone-dependent regulation of ENaC. Because TK production localizes to connecting tubule cells, the urinary side of connecting and collecting duct cells, where ENaC is expressed, is exposed to large amounts of the active enzyme. Because a novel mechanism of proteolytic activation of ENaC by locally produced serine protease has been recently proposed (11Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279: 18111-18114Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 12Vallet V. Chraibi A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. Nature. 1997; 389: 607-610Crossref PubMed Scopus (456) Google Scholar), we hypothesized that TK might be a paracrine regulator acting directly on ENaC within the ASDN. To test this hypothesis, we used a mouse model with TK gene disruption (TK–/–) to study molecular and functional expression of ENaC. Animals–The TK–/– mice were previously generated in our laboratory (6Meneton P. Bloch-Faure M. Hagege A.A. Ruetten H. Huang W. Bergaya S. Ceiler D. Gehring D. Martins I. Salmon G. Boulanger C.M. Nussberger J. Crozatier B. Gasc J.M. Heudes D. Bruneval P. Doetschman T. Menard J. Alhenc-Gelas F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2634-2639Crossref PubMed Scopus (141) Google Scholar). Type 2-bradykinin receptor knock out mice (B2–/–) mice were obtained from The Jackson Laboratory (Bar Harbor, Maine) (13Borkowski J.A. Ransom R.W. Seabrook G.R. Trumbauer M. Chen H. Hill R.G. Strader C.D. Hess J.F. J. Biol. Chem. 1995; 270: 13706-13710Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). In all experiments controls consisted in wild type littermates (WT). All the experimental procedures were performed in accordance with the French government animal welfare policy (agreement RA024647151FR). Aldosterone Infusion Study–7 TK–/– and 7 WT mice were infused continuously with aldosterone 100 μg·kg body weight–1·day–1 diluted in 0.9% NaCl and 5% Me2SO administrated by osmotic minipump (Alzet model 2004, Durect Corp., Cupertino, CA). In this particular set of experiments standard laboratory diet was supplemented with 3% Na+ and 0.4% K+. Control mice received vehicle alone. Infusion of aldosterone or vehicle was continued over a 28-day period. Physiological Studies–Animals were housed in metabolic cages and were pair-fed. After 3–5 days adaptation, urines were collected daily for electrolyte measurements. Animals were sacrificed with ketamine and xylazine (0.1 and 0.01 mg·g of body weight–1, respectively). Plasma and urine electrolytes, creatinine, and aldosterone were determined as described (14Vallet M. Picard N. Loffing-Cueni D. Fysekidis M. Bloch-Faure M. Deschenes G. Breton S. Meneton P. Loffing J. Aronson P.S. Chambrey R. Eladari D. J. Am. Soc. Nephrol. 2006; 17: 2153-2163Crossref PubMed Scopus (85) Google Scholar). Plasma renin concentration was determined by radioimmunoassay of angiotensin I generated by incubation of the plasma at pH 8.5 in the presence of an excess of rat angiotensinogen (15Menard J. Catt K.J. Endocrinology. 1972; 90: 422-430Crossref PubMed Scopus (435) Google Scholar). Plasma atrial natriuretic peptide concentration was measured by radioimmunoassay (Amersham Biosciences). Membrane Fraction Preparation–At the time of the sacrifice, kidneys were removed and cut into 5-mm slices. The renal cortex was excised under a stereoscopic microscope and placed into ice-cold isolation buffer (250 mm sucrose, 20 mm Tris-Hepes, pH 7.4) containing protease inhibitors in 4 μg/ml aprotinin, 4 μg/ml leupeptin, 1.5 μg/ml pepstatin A, and 28 μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride. Lungs were also removed, minced, and placed in the same ice-cold isolation buffer. Minced tissues were homogenized in a Dounce homogenizer (pestle A, 5 passes) followed by 10 passes through a Teflon glass homogenizer rotating at 1000 rpm. The homogenate was centrifuged at 1000 × g for 10 min, and the supernatant was centrifuged at 360,000 × g for 40 min at 4 °C. The pellet was resuspended in isolation buffer. Protein contents were determined using the Bradford protein assay (microBradford, Bio-Rad). Exosome Preparation–Urinary exosomes were prepared as previously described by Pisitkun et al. (16Pisitkun T. Shen R.F. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13368-13373Crossref PubMed Scopus (1658) Google Scholar). Briefly, urines from TK–/– and WT mice housed in metabolic cages were collected daily in tubes containing a protease inhibitor mixture (1 μg/ml leupeptin and 100 μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride) and sodium azide. A pool of 75–90 ml of urine per group was used. Urine samples were extensively vortexed immediately after they thawed. The urines were centrifuged at 17,000 × g for 15 min at 4 °C to remove whole cells, large membrane fractions, and other debris. Supernatants were centrifuged at 200,000 × g for 1 h at 4 °C to obtain a low density membrane pellet. The exosome-associated proteins isolated from the pooled urine samples were suspended in isolation solution (250 mm sucrose, 10 mm ethanolamine, pH 7.6, containing protease inhibitors in 1 μg/ml leupeptin and 100 μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride). Protein contents were determined using the Bradford protein assay (Bio-Rad). Values were normalized for urine creatinine to ensure adequate protein loading in Western blot experiments, i.e. to allow comparison of the abundance in exosome-associated proteins from urine samples of the same time for each group, as proposed by others (17Zhou H. Yuen P.S. Pisitkun T. Gonzales P.A. Yasuda H. Dear J.W. Gross P. Knepper M.A. Star R.A. Kidney Int. 2006; 69: 1471-1476Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Antibodies–Rabbit polyclonal antibodies to NHE3 and NKCC2 have been characterized previously (18Kim G.H. Ecelbarger C. Knepper M.A. Packer R.K. J. Am. Soc. Nephrol. 1999; 10: 935-942PubMed Google Scholar). Rabbit polyclonal antibodies to NaPi-2a was given by J. Biber (Zürich University, Switzerland) and has been characterized previously (19Custer M. Lotscher M. Biber J. Murer H. Kaissling B. Am. J. Physiol. 1994; 266: F767-F774PubMed Google Scholar). Chicken anti Na+,K+-ATPase polyclonal antibody was purchased from Chemicon International Inc. (Temecula, CA). Rabbit polyclonal antibodies directed to α-ENaC (amino acids 46–68), β-ENaC (amino acids 617–638), and γ-ENaC (amino acids 629–650) were raised by G. Deschenes using exactly the same peptides originally described by Masilamani et al. (20Masilamani S. Kim G.H. Mitchell C. Wade J.B. Knepper M.A. J. Clin. Investig. 1999; 104: 19-23Crossref PubMed Scopus (634) Google Scholar). All these antibodies have been described and characterized previously (21Lourdel S. Loffing J. Favre G. Paulais M. Nissant A. Fakitsas P. Creminon C. Feraille E. Verrey F. Teulon J. Doucet A. Deschenes G. J. Am. Soc. Nephrol. 2005; 16: 3642-3650Crossref PubMed Scopus (60) Google Scholar). When necessary, new batches of antibodies were tested in preliminary experiments to verify that the specificity was identical to that of the antibodies from the original batch (not shown). Immunoblot Analyses–Membrane proteins were solubilized in SDS-loading buffer (62.5 mm Tris HCl, pH 6.8, 2% SDS, 100 mm dithiothreitol, 10% glycerol, and bromphenol blue), incubated at room temperature for 30 min. Electrophoresis was initially performed for all samples on 7.5% polyacrylamide minigels (XCell SureLock Mini-cell, Invitrogen), which were stained with Coomassie Blue to provide quantitative assessment of loading, as previously described (14Vallet M. Picard N. Loffing-Cueni D. Fysekidis M. Bloch-Faure M. Deschenes G. Breton S. Meneton P. Loffing J. Aronson P.S. Chambrey R. Eladari D. J. Am. Soc. Nephrol. 2006; 17: 2153-2163Crossref PubMed Scopus (85) Google Scholar). For immunoblotting, proteins were transferred electrophoretically (XCell II Blot Module, Invitrogen) for 1.5 h at 4 °C from unstained gels to nitrocellulose membranes (Amersham Biosciences) and then stained with 0.5% Ponceau S in acetic acid to check for uniformity of protein transfer onto the nitrocellulose membrane. Membranes were first incubated in 5% nonfat dry milk in phosphate-buffered saline, pH 7.4, for 1 h at room temperature to block nonspecific binding of antibody followed by overnight at 4 °C with the primary antibody (anti-NHE3 1:1,000, anti-NaPi2a 1:20,000, anti-NCC 1:50,000, anti-NKCC2 1:5,000, anti-α-ENaC 1:3,000, anti-β-ENaC 1:20,000, anti-γ-ENaC 1:2,000, anti-Na+/K+-ATPase 1:20,000) in phosphate-buffered saline, pH 7.4, containing 1% nonfat dry milk. After four 5-min washes in phosphate-buffered saline, pH 7.4, containing 0.1% Tween 20, membranes were incubated with 1:10,000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) or peroxidase-conjugated AffiniPure donkey anti-chicken IgY (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in phosphate-buffered saline, pH 7.4, containing 5% nonfat dry milk for 2 h at room temperature. Blots were washed as above, and luminal-enhanced chemiluminescence (ECL, PerkinElmer Life Science Products) was used to visualize bound antibodies before exposure to Hyperfilm ECL (Amersham Biosciences). The autoradiography was digitized with use of a laser scanner (Epson Perfection 1650, Epson), and quantification of each band was performed by densitometry using NIH Image software. Densitometric values were normalized to the mean for the control group that was defined as 100%, and results were expressed as the mean ± S.E. For deglycosylation experiments protein samples were incubated for 1 h at 37 °C with or without peptide N-glycosidase F according to the manufacturer's instructions (Roche Diagnostics) and processed for immunoblotting as described above. To assess whether or not TK promotes γ-ENaC cleavage in vitro, renal cortical membrane fractions from TK–/– mice were prepared as described above except that proteases inhibitors were omitted. To compare their ability to promote γ-ENaC cleavage, urines from TK–/– and WT mice were first desalted against Tris buffer (0.2 m, pH 8.2) in a Centricon column (Centricon-10, cut-off 10 kDa, Millipore). The final volume of the desalted urine was one-third that of the initial volume of urine introduced in the column. Membrane fractions were incubated with desalted urines for 1.5 h at 37 °C or with purified TK from porcine pancreas (Sigma-Aldrich) before SDS-PAGE and immunoblotting with anti-γ-ENaC antibody as described above. Measurement of Alveolar Fluid Clearance in Mouse–Sodium-driven alveolar fluid clearance (AFC) was measured in vivo using an in situ non-ventilated mouse lung model, as previously described (22Fukuda N. Folkesson H.G. Matthay M.A. J. Appl. Physiol. 2000; 89: 672-679Crossref PubMed Scopus (80) Google Scholar, 23Planes C. Leyvraz C. Uchida T. Angelova M.A. Vuagniaux G. Hummler E. Matthay M. Clerici C. Rossier B. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 288: 1099-1109Crossref PubMed Scopus (70) Google Scholar). This model has been shown to give AFC values similar to those obtained with the ventilated mouse model over a 15-min period. Briefly, male or female WT or TK–/– mice aged 2–5 months were euthanized with intraperitoneal pentobarbital (250 mg/kg) and maintained at 37–38 °C using a heating pad, an infrared lamp, and an intra-abdominal monitoring thermistor. A 20-gauge venous catheter was inserted in the trachea through a tracheotomy and tightly fixed. The lungs were inflated with 100% O2 at 7 cm H2O continuous positive airway pressure throughout the experiment. Then, 10 ml/kg instillate was delivered to the lungs over 30 s through the tracheal catheter. The instillate consisted of Ringer's lactate, pH 7.4, adjusted to 325 mosmol/kg of H2O with NaCl, containing 5% bovine serum albumin, and 0.1 μCi/ml 125I-labeled albumin (Cis Bio International, Gif-sur Yvette, France) as a labeled alveolar fluid volume tracer. An alveolar fluid sample (50–100 μl) was aspirated 1 min after instillation and at the end of experiment (15 min later). The aspirates were centrifuged at 3000 × g for 10 min, and the radioactivity in supernatants was counted in duplicate. Alveolar fluid clearance (percentage fluid absorption at 15 min) was calculated from the increase in alveolar fluid albumin as AFC (%) = (Cf – Ci)/Cf × 100, where Ci and Cf represent the initial and final concentrations of 125I-labeled albumin in the aspirate at 1 and 15 min, respectively, as assessed by radioactivity measurements. In some experiments amiloride (1 mm) was added to the instillate, and AFC was measured at 15 min as described above. ENaC-mediated AFC represents the difference between total AFC values (in the absence of amiloride) and amiloride-insensitive AFC values (in the presence of amiloride). Results are presented as the means ± S.E. One-way variance analyses were performed and, when allowed by the F value, results were compared by the modified least significant difference (Statview software). p < 0.05 was considered significant. Measurement of Rectal Transepithelial Potential Difference– The mice were anesthetized with an intraperitoneal injection of Ketalar (Parke-Davis), 75 μg/g of body weight, and Rompun (Bayer, Puteaux, France), 2.3 μg/g of body weight, and placed on a heated table. A winged needle filled with isotonic saline was placed in the subcutaneous tissue of the back. A double-barreled pipette was prepared from borosilicate glass capillaries (1.0-mm outer diamater/0.5-mm inner diameter, Hilgerberg, Malsfeld, Germany) and pulled to a ∼0.2-mm tip diameter. The first barrel was filled with isotonic saline buffered with 10 mm Na+-HEPES, pH 7.2, and the second barrel with the same solution containing 25 μm amiloride. The tip of the double-barreled pipette was placed in the rectum about 3–5 mm from the skin margin. The electrical potential difference was measured between the first barrel and the subcutaneous needle, both connected to Ag+/AgCl electrodes by means of plastic tubes filled with 3 m KCl in 2% agar. The rectal potential difference (PD) was monitored continuously by a VCC600 electrometer (Physiologic Instruments, San Diego, CA) connected to a chart-paper recorder. After stabilization of the rectal PD (about 1 min), 0.05 ml of saline solution was injected through the first barrel as a control maneuver, and the PD was recorded for another 30-s period. A similar volume of saline solution containing 25 μm amiloride was injected through the second barrel of the pipette, and the PD was recorded for another minute. The amiloride-sensitive PD was calculated as the difference between the PD recorded before and after the addition of amiloride. Evaluation of Apical Na+ Entry in Isolated CCDs Microperfused in Vitro–Experiments were performed as previously described by others (24Komlosi P. Fuson A.L. Fintha A. Peti-Peterdi J. Rosivall L. Warnock D.G. Bell P.D. Hypertension. 2003; 42: 195-199Crossref PubMed Scopus (88) Google Scholar). Briefly, CCD segments were dissected from C57Bl/6 mouse, mounted on concentric pipettes, and perfused in vitro. During intracellular pH measurement experiments, the average tubule length exposed to bath fluid was limited to 300–350 μm to prevent motion of the tubule. The dissection and initial luminal and peritubular solutions were composed of 25 mm NaCl, 119 mm N-methyl-d-glucamine gluconate, 1.2 mm MgSO4, 2 mm K2HPO4, 2 mm CaCl2, 5 mm d-glucose, and 10 mm HEPES and was adjusted to a pH of 7.4. Tubules were bathed and perfused with this same solution. For the experiments CCDs were bathed in a modified solution in which NaCl was iso-osmotically replaced with N-methyl-d-glucamine gluconate to achieve an NaCl concentration of 0 mmol/liter and containing the Na+ ionophore monensin (10–5 m). This maneuver effectively eliminated the basolateral membrane as a barrier for Na+ movement. Under these conditions Na+ entry across the apical membrane is the only means of altering [Na]i. To identify principal and intercalated cells, we labeled intercalated cells by adding fluorescein-labeled peanut lectin (Vector Laboratories) to the luminal perfusate for 5 min and observed which cells were fluorescent with excitation and emission wavelengths of 440 and 530 nm, respectively. [Na]i in CCD cells was assessed with imaging-based, dual-excitation wavelength fluorescence microscopy with use of the fluorescent probe sodium-binding benzofuran isophthalate (SBFI, Molecular Probes). Tubules were loaded with 2 × 10–5 m of the acetoxymethyl ester of SBFI added to the luminal perfusate. Intracellular dye was excited alternatively at 340 and 380 nm with a 100-watt xenon lamp and a computer-controlled chopper assembly. Emitted light was collected through a dichroic mirror, passed through a 510-nm filter, and focused onto a CCD camera (ICCD 2525F, Videoscope International, VA) connected to a computer. The measured light intensities were digitized with 8-bit precision (256 gray level scale) for further analysis. For each tubule 2–4 principal cells were analyzed; the mean gray level for each excitation wavelength was calculated with the Starwise Fluo software (Imstar, Paris, France). SBFI fluorescence ratios (340/380 nm) were used as an estimator of [Na]i values. Evidence for Decreased Sodium Channel Activity in the Kidney and the Distal Colon–TK is secreted into the luminal fluid of the distal part of the ASDN, where ENaC is believed to be the limiting step for Na+ reabsorption, and a strong inverse relationship has been described between the amount of TK excreted in the urine and blood pressure in humans (3Lechi A. Covi G. Lechi C. Corgnati A. Arosio E. Zatti M. Scuro L.A. Clin. Sci. Mol. Med. 1978; 55: 51-55PubMed Google Scholar, 4Seino M. Abe K. Otsuka Y. Saito T. Irokawa N. Tohoku J. Exp. Med. 1975; 116: 359-367Crossref PubMed Scopus (51) Google Scholar). We speculated that TK might be a factor regulating ENaC activity. To test renal ENaC function in vivo, we first tested the effects of amiloride on urinary sodium excretion in TK–/– mice. Fig. 1A shows that a single dose of amiloride (subcutaneous injection of 1.45 mg·kg of body weight–1) significantly increased urinary Na+ excretion during the next 2 h after the injection in WT mice, whereas the effect of amiloride on Na+ excretion was blunted in TK–/– mice. This result indicated that renal ENaC activity was decreased in mice lacking TK production. The distal colon also expresses all three ENaC subunits, mRNA for TK has been found to be highly expressed in the colon, and abundant TK activity is measurable in the feces (6Meneton P. Bloch-Faure M. Hagege A.A. Ruetten H. Huang W. Bergaya S. Ceiler D. Gehring D. Martins I. Salmon G. Boulanger C.M. Nussberger J. Crozatier B. Gasc J.M. Heudes D. Bruneval P. Doetschman T. Menard J. Alhenc-Gelas F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2634-2639Crossref PubMed Scopus (141) Google Scholar). Thus, we hypothesized that if TK activates ENaC, this effect may be detectable in the distal colon. We tested this hypothesis in vivo by measuring amiloride-sensitive transepithelial rectal potential difference, an index of ENaC-mediated Na+ transport, in WT and TK–/– mice. As shown in Fig. 1B, the amiloride-sensitive rectal potential difference was significantly decreased in TK–/– mice compared with WT mice, indicating that, as in the kidney, TK is an activating factor of ENaC in the distal colon. Table 1 shows that TK–/– mice were able to maintain normal Na+ balance with no evidence for extracellular fluid volume depletion. Moreover, the animals were pair-fed, and hence, identical urinary excretion of Na+ and Cl– (see Table 1) indicated that they were able to achieve a steady state. Thus, although the TK–/– mice are able to excrete the full amount of NaCl taken in the diet and maintain a steady state, they do so with a concomitant decrease in amiloride-sensitive (ENaC-mediated) sodium absorption, implying that amiloride-independent Na+ absorption, presumably upstream from the collecting duct system, is increased.TABLE 1Physiological parameters from WT and TK–/– mice kept under a Na+-replete dietWT (n = 7)TK-/- (n = 7)pPlasmaRenin activity (ng of angiotensin I/h/ml)868 ± 133531 ± 71.40.049Atrial natriuretic peptide (pg/ml)325 ± 27361 ± 230.35Aldosterone (pm)555 ± 172504 ± 1020.81Protein (g/liter)48.7 ± 0.947.5 ± 0.60.32UrineUrinary volume (ml/24 h)1.28 ± 0.611.45 ± 0.250.60Creatinine (μmol/day)4.28 ± 1.755.91 ± 1.050.34Aldosterone (nmol/mmol creatinine)1.74 ± 0.201.82 ± 0.130.76Na+ (mmol/mmol creatinine)23.6 ± 3.519.2 ± 1.80.29K+ (mmol/mmol creatinine)24.0 ± 3.119.1 ± 1.20.19Cl- (mmol/mmol creatinine)40.3 ± 0.936.5 ± 1.10.07 Open table in a new tab Luminal TK Activates ENaC in Principal Cells of Cortical Collecting Duct Isolated and Microperfused in Vitro–Our preceding experiments indicate that TK–/– mice have a decreased in ENaC-mediated Na+ transport in kidney. Therefore, to confirm more directly that TK is able to activate ENaC, we next examined the effects of extracellular (luminal) TK on ENaC in CCDs isolated and microperfused in vitro. Intercalated cells were identified by adding fluorescein-labeled peanut lectin to the luminal perfusate for 5 min and observed which cells were fluorescent. [Na]i in principal cells was then assessed with use of the fluorescent probe sodium-binding benzofuran isophthalate (SBFI), as previously described (24Komlosi P. Fuson A.L. Fintha A. Peti-Peterdi J. Rosivall L. Warnock D.G. Bell P.D. Hypertension. 2003; 42: 195-199Crossref PubMed Scopus (88) Google Scholar). ENaC is the limiting step for Na+ entry into principal cells; therefore, if TK activates ENaC, [Na]i is expected to increase when TK is perfused to the luminal surface of the CCD. Accordingly, Fig. 2 shows that TK was able to increase significantly the ratio of F340/F380 SBFI fluorescence, which reflects an increase in [Na+]i, whereas no change was observed in CCDs perfused with vehicle only. Moreover, a TK-dependent increase in [Na+]i was prevented when the tubules were
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