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J. Biol. Chem., Vol. 283, Issue 8, 4602-4611, February 22, 2008

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Defective ENaC Processing and Function in Tissue Kallikrein-deficient Mice^*

Nicolas Picard¹, Dominique Eladari¹², Soumaya El Moghrabi, Carole Planès^¶, Soline Bourgeois, Pascal Houillier, Qing Wang^||, Michel Burnier^||, Georges Deschenes^**, Mark A. Knepper³, Pierre Meneton, and Régine Chambrey⁴

From the INSERM U872 (Centre de Recherche des Cordeliers), Université Paris 5, Faculté de Médecine René Descartes F-75006, Paris, Département de Physiologie, Hôpital Necker-Enfants Malades, F-75015, Paris, ^¶INSERM U773 (CRB3), Universite Paris 7, UFR de Medecine, Site Bichat, F-75018, Paris, ^||Department of Nephrology, Centre Hospitalier Universitaire Vaudois, CH-1011, Lausanne, and ^**CNRS UMR 7134, UPMC, F-75006, Paris, France and Laboratory of Kidney and Electrolyte Metabolism, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 10, 2007 , and in revised form, December 12, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 -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 -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 -ENaC, indicating that TK was able to promote -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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Tissue kallikrein⁵ (TK)⁶ 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 (1) 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 (2). 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 (3, 4), and mutant mice lacking B2 receptor also exhibit salt-sensitive hypertension (5). However, inactivation of the TK gene in the mouse does not alter blood pressure (6) 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 (7–9). 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.

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 -, β-, and -subunits, mediates Na⁺ uptake across the apical plasma membrane of principal cells and connecting tubule cells (10). 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 (11, 12), 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Animals—The TK^–/– mice were previously generated in our laboratory (6). Type 2-bradykinin receptor knock out mice (B2^–/–) mice were obtained from The Jackson Laboratory (Bar Harbor, Maine) (13). 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 (14). 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 (15). 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 x g for 10 min, and the supernatant was centrifuged at 360,000 x 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. (16). 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 x g for 15 min at 4 °C to remove whole cells, large membrane fractions, and other debris. Supernatants were centrifuged at 200,000 x 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 (17).

Antibodies—Rabbit polyclonal antibodies to NHE3 and NKCC2 have been characterized previously (18). Rabbit polyclonal antibodies to NaPi-2a was given by J. Biber (Zürich University, Switzerland) and has been characterized previously (19). Chicken anti Na⁺,K⁺-ATPase polyclonal antibody was purchased from Chemicon International Inc. (Temecula, CA). Rabbit polyclonal antibodies directed to -ENaC (amino acids 46–68), β-ENaC (amino acids 617–638), and -ENaC (amino acids 629–650) were raised by G. Deschenes using exactly the same peptides originally described by Masilamani et al. (20). All these antibodies have been described and characterized previously (21). 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 (14). 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--ENaC 1:3,000, anti-β-ENaC 1:20,000, anti--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 -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 -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--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 (22, 23). 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 x 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 x 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 (24). 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 x 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (3, 4). 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 (6). 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.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Effects of TK disruption on ENaC function in the kidney and colon. A, effect of amiloride injection on urinary Na+ excretion in TK–/– and WT mice. Mice kept under a normal Na+ diet (0.3% Na+ in food) were subcutaneously injected with amiloride (1.45 mg/kg of body weight). Urines from TK–/– (filled square) and WT mice (filled triangle) were collected over 2 days from 9 to 11 a.m., 11 a.m. to 2 p.m., 2 to 6 p.m., and 6 p.m. to 9 a.m.; amiloride was injected at 9 a.m. the second day. UNa+/Ucreat represents the urinary Na+ concentration in mM normalized to the urinary concentration of creatinine in mM. Values are the means ± S.E. from 5 to 9 determinations in each group. p < 0.05 (*) and p < 0.01 (**) versus time controls; #, p < 0.01 versus TK–/– mice. B, diurnal variations in amiloride-sensitive rectal PD in TK–/– mice. In vivo measurements of amiloride-sensitive rectal PD in TK–/– (white bars) and WT (black bars) mice were performed in the morning (1012 a.m.) and afternoon (46 p.m.). The data show a significant decrease in amiloride-sensitive sodium transport in the colon of TK–/– mice (n = 4 to 6 mice per group). *, p < 0.05.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Time course of SBFI fluorescence, an estimate of [Na+]i, in principal cells of CCDs isolated and microperfused in vitro. CCDs isolated from WT mice were perfused with either TK (10 µg/ml) in the absence (filled circles) or the presence of 10–5 M amiloride (asterisk) or perfused with vehicle alone (open circles). After 10 min of TK perfusion, SBFI fluorescence increased significantly in TK treated tubules (p < 0.05 versus controls), reflecting a rise in [Na+]i, whereas no change occurred in CCDs perfused with vehicle. Moreover, 10–5 M amiloride in the luminal fluid prevented the TK-induced rise in [Na+]i, demonstrating that this effect reflects ENaC activation by TK (n = 3 in each group).

-ENaC Processing Is Impaired in TK^–/– Mice—Because TK is a serine protease, it is possible that TK-induced ENaC activation occurs through ENaC proteolysis. Thus, we next examined ENaC-related polypeptides by Western blot. As shown in Fig. 3, antibodies to the C terminus of -ENaC detect a major band at 85 kDa as well as a minor 80-kDa band. An additional broad band centered around 70 kDa was detected in WT mice but not in TK^–/– mice. This 70-kDa band is believed to originate from the proteolytic cleavage of the N-terminal end of the 85-kDa form and to be related to aldosterone-dependent activation of ENaC (20). When renal samples from WT mice were deglycosylated with peptide N-glycosidase and immunoblotted using anti- ENaC, the 85-kDa band shifted to 72 kDa, and the broad 70-kDa band shifted to a major 50-kDa band and a weaker 60-kDa band (Fig. 4A). No change in protein abundance of either the and β subunits of ENaC or other Na⁺ transporter expressed along the nephron was detected (Fig. 3, Table 2).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Immunoblot of renal cortex membrane fractions from TK–/– and WT mice with antibodies to -, β-, and -subunit of ENaC. The 70-kDa -ENaC polypeptides were undetectable in membrane fractions from TK–/– mice, whereas and β-subunit-related polypeptides were unchanged. Each lane was loaded with a protein sample from a different mouse. Equal loading was confirmed by parallel Coomassie-stained gels.

View larger version (44K): [in this window] [in a new window]   FIGURE 4. Glycosylation pattern of -ENaC protein in mouse renal cortex. Protein samples (A, membrane fractions from TK–/– and WT mice under basal conditions and aldosterone-infused TK–/– and WT mice renal cortex; B, membrane fractions from aldosterone-infused TK–/– and WT mice renal cortex) were incubated with or without N-glycosidase F (PNGase) for 1 h at 37 °C before SDS-PAGE and immunoblotting for -ENaC.

View larger version (36K): [in this window] [in a new window]   FIGURE 5. Tissue Kallikrein promotes -ENaC cleavage in vitro. Panel A, renal membrane fractions from TK–/– mice were incubated with TK, and apparition of the 70-kDa form of -ENaC was studied by immunoblotting. Membrane proteins were mixed with either 0, 30, or 100 µg/ml TK purified from porcine pancreas and incubated for 15 min at 37 °C. Panel B, densitometric analyses of data showing the appearance of the 70-kDa form after treatment with TK. Panel C, renal membrane fractions from TK–/– mice were incubated with desalted and concentrated urines from WT or TK–/– mice for 1.5 h at 37 °C. The apparition of the 70-kDa form of -ENaC was studied by immunoblotting.

View larger version (55K): [in this window] [in a new window]   FIGURE 6. Abundance of ENaC subunits and NCC by immunoblotting in urinary exosomes from TK–/– and WT mice normalized to urine creatinine and kidney cortex from wild type mouse. Lanes, from left to right, were loaded with protein samples from wild type mouse kidney cortex (lane 1), urinary exosomes from wild type mice (lane 2), and urinary exosomes from TK–/– mice (lane 3). Immunoblots are representative of three independent experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 7. Abundance of -ENaC protein by immunoblotting in membrane fractions from kidney cortex of B2–/– and WT mice. Panel A, immunoblot of renal cortex membrane fractions from TK–/– and WT mice. Each lane was loaded with a protein sample from a different mouse. Equal loading was confirmed by parallel Coomassie-stained gels. Panel B, densitometric analyses of immunoblots showing that the abundance of the 70-kDa form is significantly increased in B2–/– mice. *, p < 0.05 versus WT mice.

Evidence for Unchanged Sodium Channel Activity in Lung— In contrast to the kidney and the colon, lung alveolar epithelial cells express ENaC but do not secrete TK; therefore, we hypothesized that ENaC processing in the lung is TK-independent. Fig. 8A shows that amiloride-sensitive alveolar fluid clearance, used as an estimate of ENaC-mediated Na⁺ transport (23), was not significantly different in TK^–/– mice compared with WT mice. Fig. 8B shows that the biochemical profile of -ENaC in the lung appears as two major bands, one at 85 kDa, similar to that observed in the kidney, and another one resolving at 75–70 kDa. These bands were no longer detected when anti--ENaC was preincubated with the peptide antigen (data not shown). There was no significant difference in the abundance of the 85-kDa -ENaC (102 ± 5 versus 100 ± 12% in TK^–/– and WT mice, respectively) and of the 75–70-kDa -ENaC (103 ± 6 versus 100 ± 8% in TK^–/– and WT mice, respectively). Deglycosylation with peptide N-glycosidase gave a pattern similar to that seen in renal samples from WT mice except that the size of the major fragment was 60 kDa (data not shown).

View larger version (41K): [in this window] [in a new window]   FIGURE 8. Analyses of -ENaC polypeptides and of ENaC function in the lung. Panel A, effect of TK knock-out on sodium-driven alveolar fluid clearance in mice. Sodium-driven AFC was measured over a 15-min period in WT and TK–/– mice aged 2–5 months at 37 °C using an in situ non-ventilated model in which the airspace was instilled with an iso-osmolar Ringer's lactate solution containing 125I-labeled albumin as a volume marker, as described under "Experimental Procedures." Experiments were performed either in the absence (total AFC, black bars) or in the presence of 1 mM amiloride (white bars) in the instillate. Results are expressed as percentage fluid absorption at 15 min and represent the means ± S.E. of 8–9 mice in each group for total AFC experiments and 4 mice in each group for amiloride experiments. Panel B, abundance of -ENaC protein by immunoblotting in membrane fractions from lung of TK–/– and WT mice. Each lane was loaded with a protein sample from a different mouse. Equal loading was confirmed by parallel Coomassie-stained gels. No significant differences in the abundance levels of the 85-kDa band and the 75–70-kDa band were observed between WT and TK–/– mice. *, significantly different from corresponding total AFC value (p < 0.05). NS, not significantly different.

View larger version (44K): [in this window] [in a new window]   FIGURE 9. Effect of TK knock-out on -ENaC protein in mice undergoing dietary Na+ restriction or chronic aldosterone infusion. Immunoblots for -ENaC of membrane fractions from kidney cortex of TK–/– and wild type mice under Na+ restricted diet (A) or after chronic aldosterone administration (B). Each lane was loaded with a protein sample from a different mouse. Equal loading was confirmed by parallel Coomassie-stained gels. The asterisk indicates a protein sample from a mouse that had not correctly received aldosterone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ENaC is a heteromultimeric protein composed or three related subunits , β, and . All subunits have an intracytoplasmic N and C terminus and two membrane spanning domains connected by a large extracellular loop. ENaC activity can be regulated by changes in membrane protein abundance of the channel or by changes in single-channel open probability. Recently, it has been proposed that proteolytic processing of and subunits by several serine proteases increases ENaC open probability (11, 12, 27–29). Conversely, non-processed ENaC have been demonstrated to be almost inactive when expressed in cell cultures. In this model extracellular trypsin stimulates ENaC, whereas extracellular serine protease inhibitors, such as aprotinin and bikunin, have been shown to decrease ENaC activity (12, 29–32). However, despite this evidence that proteolytic processing of ENaC stimulates channel activity, the molecular identity of the protease that could mediate this effect in vivo is still uncertain. As stated above, trypsin can activate ENaC. Elastase, a serine protease naturally produced by neutrophil polynuclear has also been proposed to activate ENaC in vivo during pulmonary inflammatory states (28). More recently, direct evidence for exogenous cleavage of -ENaC and activation at the cell surface by elastase in Xenopus oocytes experiments has been provided by Harris et al. (33). However, trypsin and elastase are not very good candidates for in vivo physiological regulation of ENaC in the kidney because they are normally not expressed and secreted in this organ. Furin, a proprotein convertase involved in many physiological or pathophysiological processes, including activation of hormones and growth factors to bacterial toxins and viral glycoproteins processing, is also able to cleave and activate ENaC in vitro (11, 34). A family of serine proteases has been identified that could cleave and activate ENaC, including CAP1, CAP2, CAP3, and TMPRSS3 (12, 23, 35–38). Importantly, it has been shown that aldosterone modifies urinary excretion of prostasin, the human ortholog of CAP1 (39), suggesting that prostasin could be the link between aldosterone and proteolytic activation of ENaC in the kidney. Unfortunately, gene disruption of furin or CAP1 are lethal, rendering difficult direct assessment of their physiological role in ENaC regulation (40, 41). It is also possible that proteolytic activation of ENaC involves more than one effector. This is supported by the observation that the proteolytic processing of -ENaC by furin seems to occur intracellularly, whereas trypsin, elastase, or the CAPs are believed to act extracellularly. Moreover, it has recently been demonstrated that furin or prostasin is able to produce different polypeptides from -ENaC (42).

Here we report that a serine protease promotes -ENaC cleavage in vivo and in vitro (see Figs. 1A and 5) and that this proteolytic modification correlates with the activation of ENaC. Active TK excretion is positively correlated with miner-alocorticoid levels (43, 44). Interestingly, it has also been shown that the protein abundance of the 70-kDa form of -ENaC (the truncated form which disappears in TK^–/– mice) also correlates with circulating aldosterone level (20). Therefore, it is tempting to conclude that TK is responsible for part of the aldosterone-dependent processing/activation of the channel. However, it is important to state that it is not possible to conclude that TK is directly responsible for the cleavage itself (i.e. that TK physically interacts with and cleaves -ENaC). Indeed, in our in vitro experiments we used membrane fractions from TK^–/– mice. This material does not contain TK, but it probably expresses several other proteases, particularly several members of the CAP family that are membrane-bound. Therefore, it is possible that the action of TK on -ENaC is indirect. For example, it is conceivable that TK activates a CAP-dependent signaling cascade, leading ultimately to ENaC processing as proposed by B. Rossier recently (45).

Because the TK^–/– mice did not exhibit gross alterations in sodium balance or extracellular fluid regulation, it could be also argued that TK does not play a substantial physiological role under normal circumstances. However, because regulation of Na⁺ reabsorption in the kidney is critical for extracellular fluid volume and blood pressure regulation, the mechanisms for regulation of Na⁺ reabsorption are highly redundant. That is, animals can generally compensate for defective regulation at one renal tubule segment by increasing absorption at another renal tubule segment, lowering blood pressure or reducing glomerular filtration rate to come into Na⁺ balance. Thus, in the present study altered regulation at the level of ENaC is likely to be compensated for. One example of this is seen in mice in which the thiazide-sensitive NaCl cotransporter is responsible for 5–10% of sodium reabsorption under normal circumstance (46). These mice appear healthy and are normal with respect to plasma electrolyte concentrations, serum aldosterone levels, and blood pressure. Moreover, NCC^–/– mice retain Na⁺ as well as wild-type mice when fed a Na⁺-depleted diet (47). In contrast, these mice show abnormalities in homeostasis of other ions including Ca²⁺ (47) and K⁺ (48). One other possible explanation is that the TK gene inactivation does not only disrupt TK production but also impairs local bradykinin production. Inhibition of bradykinin production is expected to limit natriuresis, whereas TK disruption is expected to limit ENaC activation. Therefore, the phenotype arising from both effects might be very limited. Finally, in the absence of TK some proteolytic activation of the channel can still occur and produce several different polypeptides that are presumably active (see Fig. 9). The role of furin or CAPs in this compensation requires further investigation. For example, CAP could permit the cleavage of the 85-kDa -ENaC form and, therefore, activation of ENaC in TK^–/– mice with elevated circulating aldosterone. Based on the size of the fragments obtained after deglycosylation of the cleaved form of -ENaC (60 and 50 kDa), which are recognized by the anti--ENaC antibody directed against the C terminus of -ENaC, the proteolytic cleavage seems to occur at two different sites in the early portion of the extracellular loop of the -ENaC which are approximately 100 amino acids apart. Deglycosylation of renal samples from either control or aldosterone-treated WT mice showed that the 70-kDa protein shifted to a major 50-kDa protein (Fig. 2). Deglycosylation of kidney samples from aldosterone-infused TK^–/– mice showed that the broad 75–70-kDa protein shifted to a major 60-kDa protein and a weaker 50-kDa protein (Fig. 2). Membrane samples from lung, an organ not expressing TK (6), showed a similar pattern after deglycosylation. These results suggest that the renal 70-kDa -ENaC could result from the proteolytic cleavage of the 85-kDa -ENaC form by the TK itself (or at least dependent on TK), whereas the 75–70-kDa broad protein seen in kidney sample from TK^–/– mice with elevated circulating aldosterone or lung sample from either WT or TK^–/– mice could result from the proteolytic cleavage of the 85-kDa -ENaC by another serine protease such as CAPs or furin. We also report that ENaC is functionally normal in lung alveoli of TK^–/– mice, confirming that ENaC in the lung can be processed by another protease than TK. These observations highlight the fact that TK-dependent activation of ENaC may be tissue-specific.

In conclusion, this study identified an anti-natriuretic effect of TK, and we propose that TK is a physiological activator of ENaC that belongs to a complex cascade of protease that is required for full stimulation of Na⁺ transport within the ASDN. The data from our model of TK inactivation strongly suggest that the decrease in renal TK production that has been found repeatedly to be associated with high blood pressure in humans is rather a consequence than a cause of this disease.

	FOOTNOTES

 
^* This work was supported in part by the INSERM and by the Société de Néphrologie (Ph.D. support to N. P. and S. E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work and share first authorship.

³ Supported by the intramural budget of the NHLBI, National Institutes of Health Project HL-001285.

² To whom correspondence may be addressed: INSERM Unité 872, Centre de Recherche des Cordeliers, 15 rue de l'Ecole de médecine, 75006, Paris, France. Tel.: 33-1-44-41-37-18; Fax: 33-1-44-41-37-17; E-mail: eladari{at}ccr.jussieu.fr. ⁴ To whom correspondence may be addressed: INSERM Unité 872, Centre de Recherche des Cordeliers, 15 rue de l'Ecole de médecine, 75006, Paris, France. Tel.: 33-1-44-41-37-18; Fax: 33-1-44-41-37-17; E-mail: chambrey{at}ccr.jussieu.fr.

⁵ In 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.

⁶ The abbreviations used are: TK, tissue kallikrein; PD, potential difference; SBFI, sodium-binding benzofuran isophthalate; CCD, cortical collecting duct; ASDN, aldosterone-sensitive distal nephron; WT, wild type; AFC, alveolar fluid clearance; CAP, channel-activating protease.

	ACKNOWLEDGMENTS

 
We thank Bernard Rossier for critical reading of the manuscript and helpful discussion.

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