 |
||||
| 2008 JASN IMPACT FACTOR 7.505 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2008 JASN IMPACT FACTOR 7.505 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
BASIC RESEARCH |
* Physiology and Pharmacology, Institute of Medical Biology, University of Southern Denmark, Department of Nephrology Y, Odense University Hospital, Odense C, and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark; and Department of Cellular and Molecular Physiology, University of Erlangen-Nuremberg, Erlangen, Germany
Correspondence: Dr. Claus Bistrup, Department of Nephrology Y, Odense University Hospital, Sdr. Boulevard 29, DK-5000 Odense C, Denmark. Phone: +4565411762; Fax: +4565413452; E-mail: claus.bistrup{at}ouh.regionsyddanmark.dk
Received for publication April 8, 2008. Accepted for publication September 2, 2008.
 |
Abstract |
|---|
 Top Abstract IntroductionRESULTSDISCUSSIONCONCISE METHODSNOTE ADDED IN PROOFDISCLOSURESREFERENCES |
|---|
subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC. |
Introduction |
|---|
|
TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSNOTE ADDED IN PROOFDISCLOSURESREFERENCES |
|---|
, β, .3 ENaC activity is regulated by hormones, such as aldosterone and vasopressin (AVP)4,5; however, adrenalectomized rats and AVP-deficient Brattleboro rats are capable of developing nephrotic syndrome,1,6 and nephrotic patients do not consistently display elevated levels of sodium-retaining hormones,7,8 suggesting that renal sodium hyper-reabsorption is independent of systemic factors. Consistent with this, sodium retention is confined to the proteinuric kidney in the unilateral puromycin aminonucleoside (PAN) nephrotic model.2,9,10
There is evidence that proteases contribute to ENaC activation by cleaving the extracellular loops of the - and -subunits.11–13 Proteolytic activation of ENaC by extracellular proteases critically involves the cleavage of the subunit,14–16 which probably leads to the release of a 43-residue inhibitory peptide from the ectodomain.17 Both cleaved and noncleaved channels are present in the plasma membrane,18,19 allowing proteases such as channel activating protease 1 (CAP1/prostasin),20 trypsin,20 chymotrypsin,21 and neutrophil elastase22 to activate noncleaved channels from the extracellular side.23,24 We hypothesized that the defective glomerular filtration barrier in nephrotic syndrome allows the filtration of ENaC-activating proteins into the tubular fluid, leading to stimulation of ENaC. The hypothesis was tested in the PAN nephrotic model in rats and with urine from patients with nephrotic syndrome.
|
RESULTS |
|---|
|
TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSNOTE ADDED IN PROOFDISCLOSURESREFERENCES |
|---|
|
Plasmin in Nephrotic Urine Stimulates ENaC Activity
We hypothesized that a serine protease may be present in nephrotic urine and may directly activate ENaC in the plasma membrane. Whole-cell current did not change in single M-1 cells when exposed to urine from control rats, whereas urine collected on day 5 after PAN injection yielded a 5.1 ± 2.0-fold increase in the observed inward current that was prevented in the presence of 2 µM amiloride (control urine –55 ± 12 pA versus nephrotic urine –231 ± 53 pA at –150 mV holding potential; n = 4; P < 0.05; Figure 2, A and B). This stimulatory effect was observed within 30 to 60 s after exposure of the M-1 cells to nephrotic urine, and it was prevented by aprotinin, an inhibitor of serine proteases (Figure 2B). Xenopus laevis oocytes heterologously expressing ENaC confirmed the stimulatory effect of nephrotic urine samples on amiloride-sensitive ENaC whole-cell currents (
Iami; Figure 2C) compared with control oocytes preincubated in ND96 bath solution or compared with oocytes preincubated in heat-inactivated nephrotic urine samples. The stimulatory effect of a 30-min exposure to nephrotic urine was similar to that of a 5-min exposure to chymotrypsin.21 These findings suggest that nephrotic urine contains a serine protease, which can activate ENaC.
|
Aprotinin-affinity precipitation of nephrotic urine effectively removed serine protease activity and abolished the ability to activate ENaC current (Figure 3A), whereas total protein concentration was not changed (35.8 versus 34.0 mg/ml for nephrotic urine and aprotinin-affinity supernatant, respectively). The aprotinin-affinity precipitate was highly enriched in serine protease activity compared with nephrotic urine (30.80 ± 0.60 versus 0.20 ± 0.08 A405 nm/h per mg protein, respectively) and stimulated ENaC activity (Figure 3A). Ion exchange chromatography of the precipitate followed by protease activity screening identified fractions with high activity (Figure 3B). Peak fractions corresponding to elution volume 29 to 31 ml were pooled and subjected to repeated aprotinin-affinity precipitation. The precipitate was separated by gel electrophoresis, which yielded two distinct bands with a molecular weight of 40 and 80 kD (Supplemental Figure 3A). Matrix-assisted laser desorption/ionization time of-flight (MALDI-TOF) mass spectrometry identified the proteins as plasmin fragments and plasmin, respectively (Figure 3C, Supplemental Table 2). Zymography using plasmin but not plasminogen exhibited a pattern similar to the proteolytic activity observed with nephrotic urine (Supplemental Figure 3B). Pure plasmin activated a sodium current in M-1 cells that was inhibited by amiloride and by knockdown of the ENaC subunit using small interfering RNA (siRNA; Figure 3, D and E). The protease activity of nephrotic urine was inhibited by the serine protease inhibitor aprotinin, the specific plasmin inhibitors Pefabloc PL, and 2-antiplasmin (Supplemental Figure 4A). The selective plasmin inhibitors prevented the ability of nephrotic urine to activate ENaC currents in single M-1 cells when they were added to the urine samples before exposing the cells to the samples (Figure 4). Thus, plasmin is the dominant protease in nephrotic urine responsible for ENaC activation.
|
|
|
A405nm/h/ml without and with amiloride). Thus, amiloride did not inhibit plasmin per se, whereas it inhibited urinary formation of plasmin from plasminogen.
uPA-Mediated Conversion of Plasminogen to Plasmin Is Required for ENaC Activation
In Xenopus laevis oocytes heterologously expressing rat ENaC, preincubation of the oocytes with uPA alone had only a marginal stimulatory effect on the Iami, and exposure to plasminogen had no significant effect. In contrast, the combination of plasminogen and uPA resulted in a significant increase in Iami, similar to the increase observed with chymotrypsin (Figure 6A). This indicates that uPA-mediated generation of plasmin is required for ENaC activation. To investigate whether ENaC activation is associated with ENaC cleavage at the cell surface, we used a ENaC construct with a C-terminal V5 tag to detect biotinylated cell surface ENaC fragments by Western blot. Exposure of the oocytes to a combination of uPA and plasminogen yielded a ENaC fragment with a molecular weight of approximately 67 kD (Figure 6B). We observed similar fragments when trypsin or chymotrypsin was used. As previously shown,15,16,26 the 76-kD band corresponds to a cleavage product that arises from ENaC cleavage at its putative furin cleavage site when ENaC is coexpressed with the - and β-subunits. Full-length ENaC migrates at approximately 87 kD and is readily detectable in whole-cell lysate but not at the cell surface (Figure 6B). Full-length ENaC is the predominant band detected in oocytes expressing ENaC in the absence of the - and β-subunits (Figure 6B).
|
Plasmin Stimulation Leads to Release of a Peptide from the Extracellular Domain of ENaC
Proteolytic activation of ENaC is thought to involve dual cleavage of its -subunit, which results in the release of a 43-residue inhibitory peptide from its extracellular domain.17 To examine this in live cells, we inserted a hexahistidine tag in a ENaC construct expression vector between the cleavage sites for furin and prostasin (Figure 7). The tag binds the extracellular fluorophore NTA-Atto550. M-1 cells transfected with the tagged construct display strong fluorescence when exposed to NTA-Atto550, whereas wild-type cells do not (Figure 7). Pretreatment of transfected cells with plasmin or nephrotic urine abolished the ability of cells to respond to NTA-Atto550 exposure. This indicates effective removal of inhibitory peptide from ectodomain ENaC by plasmin in live cells (Figure 7).
|
2-antiplasmin (5 µM) or amiloride (2 µM) abolished the ability of nephrotic urine to stimulate whole-cell currents in M-1 cells (Figure 8B). Urine zymograms displayed intense proteolytic activity with apparent molecular weight of 75 to 80 kD (Figure 8C). Serine protease activity sensitive to plasmin inhibitors was present in patient urine samples but not in urine from four age- and gender-matched control subjects (Figure 8D). Western blots for plasminogen and plasmin displayed several bands in nephrotic but not in control urine (Figure 8E). Immunohistochemical staining of human nephrectomy specimens showed uPA predominantly in the CCD (Figure 8F). Western blotting with human kidney homogenates confirmed expression of uPA in kidney cortex (Figure 8F).
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSNOTE ADDED IN PROOFDISCLOSURESREFERENCES |
|---|
ENaC subunit. Urine from patients with nephrotic syndrome contains active plasmin, which is the dominant serine protease in nephrotic urine able to activate ENaC. On the basis of the temporal association between appearance of plasmin in urine and primary, renal ENaC-mediated sodium hyper-reabsorption, the findings provide a putative novel mechanistic link between damage to the glomerular filtration barrier, proteinuria, and stimulation of sodium reabsorption in distal tubules and collecting ducts in nephrotic syndrome. Simultaneous with the onset of proteinuria at day 3 after PAN injection, the cumulated sodium balance changed from negative to positive, and the renin-angiotensin-aldosterone system was suppressed. In this phase, mineralocorticoid receptor blockade, adrenalectomy, angiotensin II receptor blockade, or angiotensin converting enzyme blockade had no effect on renal sodium output or development of ascites.2,29 In agreement with previous data,29 treatment of nephrotic rats with amiloride in the period with positive sodium balance increased sodium excretion and normalized sodium balance, suggesting that the increased sodium reabsorption occurs through ENaC in the aldosterone-sensitive part of the distal nephron. Consistent with this, there is increased expression and activity of the Na,K-ATPase in isolated CCD from rats with nephrotic syndrome.29
The onset of proteinuria in PAN nephrotic syndrome coincided with the shift from a negative to a positive sodium balance. A correlation between proteinuria and an increase in sodium reabsorption has also been observed in the HgCl2 nephropathy model in rats.1 Taken together with the reports of extracellular activation of ENaC by proteases,11–13,20 these observations led us to hypothesize that nephrotic urine contains an ENaC-activating factor. We found that PAN nephrotic urine stimulated amiloride-sensitive sodium currents dependent on serine protease activity that could be heat inactivated.18,20 We identified plasmin in nephrotic urine as the dominant serine protease responsible for activation of sodium currents. Only the combination of plasminogen and uPA, which allows formation of plasmin, stimulates ENaC-mediated short circuit current in M-1 cell monolayers and amiloride-sensitive whole-cell current in ENaC-expressing Xenopus laevis oocytes. The large plasmin-induced increase in ENaC currents in Xenopus laevis oocytes was similar to that induced by chymotrypsin, trypsin, or neutrophil elastase.15,21 Moreover, the combined application of uPA and plasminogen led to the appearance of a ENaC cleavage product at the plasma membrane, which was of a similar size as the fragment observed with trypsin, chymotrypsin, and human neutrophil elastase.16 Consistent with this, plasmin removed a labeled inhibitory peptide region from the extracellular domain of the ENaC subunit in live cells. These findings indicate that ENaC activation by plasmin involves proteolytic cleavage in the extracellular loop of the channel's -subunit.
Plasmin and plasminogen were present in nephrotic urine, and the nephrotic urine displayed significantly increased serine protease activity compared with control urine; however, plasma serine protease activity was not different between control and nephrotic rats, and only plasminogen was detected by Western blotting in plasma from control rats. This indicates that plasmin is unlikely to be filtered in large amounts and that the plasminogen-plasmin conversion occurs after glomerular filtration. A candidate for renal tubular plasminogen activation is uPA.30,31 In agreement with previous findings,30 we found uPA expressed at the apical surface of CCD. Western blotting confirmed expression of uPA in rat kidney cortex. Amiloride, in addition to being a potent inhibitor of ENaC, is known to inhibit uPA effectively,25 and treatment of nephrotic rats with amiloride abolished the activation of plasminogen. Tubular uPA is a likely candidate to convert plasminogen to plasmin in nephrotic syndrome.
Human urine samples obtained from patients with nephrotic urine displayed plasmin(ogen) immunoreactivity in Western blots, gelatinase activity in zymograms, and serine protease activity by enzyme assay, whereas control urine samples all were negative. Human nephrotic urine stimulated sodium currents in single M-1 cells, and the stimulation was sensitive to inhibitors of plasmin, which indicates that plasmin is the main ENaC activator also in human nephrotic urine. Furthermore, uPA immunoreactivity was found predominantly in the cortex of human kidney samples, consistent with previous findings.32 The presence of active plasmin in urine and the ability of nephrotic urine to activate ENaC are, thus, not specific for the animal model, and the established beneficial effect of amiloride in nephrotic syndrome may include a hitherto unrecognized target, uPA. Thus, our results derived from the rat model of nephrotic syndrome may be clinically relevant, and it can be speculated that the proposed mechanism may promote sodium retention in pathologic conditions with proteinuria. It is not known to what extent endogenous proteases constitutively activate ENaC in vivo under various physiologic and pathologic conditions. The activity of endogenous proteases and protease inhibitors may be hormonally regulated. In humans, states of low plasma aldosterone seem to be associated with decreased urinary prostasin excretion,33 whereas high concentrations of aldosterone have been shown to increase the expression of prostasin34 and to downregulate the expression of the endogenous serine protease inhibitor, nexin 1 (PN-1), in cultured M-1 cells.35 It was recently shown that rats fed a 1% sodium diet expressed both full-length and cleaved ENaC at the plasma membrane, whereas Na-depleted or aldosterone-treated rats mainly expressed the cleaved subunit.36 In agreement, we37 and others36 recently demonstrated that trypsin can activate ENaC in microdissected mouse and rat distal nephron and that the stimulatory effect of trypsin was more pronounced in tubules from animals maintained on a standard-sodium diet than animals maintained on a low-sodium diet. These findings demonstrate that ENaC can be activated by extracellular proteases in the native renal tubule under normal physiologic conditions and that the degree of proteolytic preactivation of ENaC by endogenous proteases will determine the size of the additional stimulatory effect of proteases such as plasmin occurring in the urine under pathophysiologic conditions.
In summary, our study introduces the novel concept that a leaky glomerular filtration barrier allows filtration of proteases or precursors of proteases with the ability to activate ENaC. Plasmin is generated in tubular fluid from filtered plasminogen by amiloride-sensitive uPA and is the dominant serine protease in nephrotic urine. Plasmin activates ENaC in vitro by proteolytic cleavage of the -subunit. We do not know whether plasmin-induced cleavage of ENaC is a direct effect of plasmin on the channel or the result of plasmin-mediated activation of another membrane-anchored protease or protease cascade. It may be speculated that ENaC stimulation by plasmin contributes to hitherto unexplained primary renal NaCl reabsorption in nephrotic syndrome.
|
CONCISE METHODS |
|---|
|
TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSNOTE ADDED IN PROOFDISCLOSURESREFERENCES |
|---|
Food, Urine, and Fecal Analysis
Determination of sodium content in food, urine, and feces was as described previously.38 Daily sodium balance was calculated as intake minus fecal and urinary output. The magnitude of accumulated sodium balance was calculated as area under the curve from days 0 through 8.
Blood Analysis
The plasma concentration of sodium and urea and the plasma osmolality were determined on the day of decapitation (day 8 after PAN or vehicle injection). Plasma renin, aldosterone, and AVP concentrations were determined as described previously.38,43,44
Purification of ENaC-Activating Proteins
Aprotinin (USB, Cleveland, OH) was coupled to CNBr-activated Sepharose 4B (Amersham Bioscience, Hillerod, Denmark) according to the manufacturer's protocol and added to the nephrotic urine. The beads were pelleted and washed thoroughly. Bound proteins were eluted and loaded onto a Resource Q column (Amersham Bioscience) equilibrated with 20 mM Tris (pH 7.4). The column was eluted with a linear gradient from 0 to 1 M NaCl. Fractions were collected and assayed for serine protease activity. Fractions with high serine protease activity were subjected to aprotinin-affinity precipitation.
Serine Protease Assay and Zymography
We measured serine protease activity with the chromogenic substrate S-2222 (Chromogenix, Frederiksberg, Denmark) by spectrophotometrically (VersaMax microplate reader; Molecular Devices, Sunnyvale, CA) assessing the change in absorbance at 405 nm. Urinary protease activities were examined by zymography (Invitrogen, Tåstrup, Denmark) according to the manufacturer's protocol.
Mass Spectrometry
SDS-PAGE, silver staining, and tryptic in-gel digestion were done as described previously.45 Mass spectra of the tryptic peptides were obtained with the Ultraflex II TOF/TOF system from Bruker (Bremen, Germany). The proteins were identified by searching against the NCBI nonredundant protein database using Mascot (Matrix Science, http://www.matrixscience.com).
Whole-Cell Patch Clamp of Single M-1 Cells
M-1 cells were obtained from ATCC (Boras, Sweden) and maintained in DMEM:F12 (Life Technologies, Tåstrup, Denmark) supplemented with 5% FCS (Life Technologies) and 5 µM dexamethasone (Sigma, St. Louis, MO) at 37°C/5% CO2. For patch-clamp experiments, M-1 cells grown to confluence in 25-cm2 flasks (Nunc, Roskilde, Denmark) were trypsinized and seeded onto coverslips in DMEM:F12 (Life Technologies) supplemented with 5 µM dexamethasone (Sigma). The cells were incubated at 37°C/5% CO2. Patch-clamp experiments were conducted on single M-1 cells 24 to 48 h after seeding the cells. Details regarding the patch-clamp experiments are given in the online supplemental material. For transepithelial studies, M-1 cells were seeded onto permeable Millicell-HA culture plate inserts (Millipore GmbH, Schwalbach, Germany) and were grown to confluence in PC1 culture medium (Lonza, Verviers, Belgium) as described previously.27
Ussing Chamber
Experiments were essentially performed as described previously27 but with slightly modified Ussing chambers to reduce the volume needed for apical solution exchanges. In confluent M-1 cells, application of trypsin to the apical side had little effect on ENaC-mediated short-circuit current,46 probably because the activity of endogenous proteases is high under our culture conditions, resulting in a high baseline activity of ENaC close to that observed in fully stimulated collecting duct principal cells in native tissue.28 As recently reported, pretreatment of M-1 cells with a furin inhibitor largely augmented the stimulatory effect of trypsin on ENaC currents, whereas pretreatment with aprotinin had little effect.37 Thus, to minimize ENaC activation by endogenous proteases, the confluent M-1 cells grown on filters were preincubated for 6 to 8 h before the experiment with the furin inhibitor dec-RVKR-cmk (decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone from Calbiochem/Merck Biosciences GmbH, Schwalbach, Germany) which was added to the apical bath solution. A 10 mM stock solution of dec-RVKR-cmk was prepared in DMSO and was diluted to a final concentration of 40 µM.
Identification of ENaC Transcripts in M-1 Cells
RNA was extracted from M-1 cells in accordance with the manufacturer's protocol (Qiagen Mini Kit; Qiagen, Ballerup, Denmark) and reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad, Copenhagen, Denmark). ENaC subunit cDNA were amplified with rat ENaC primers that also annealed to mouse sequence (Supplemental Table 4).
Western Blotting
Kidneys were homogenized as described previously.44 Plasma, urine, or homogenate samples were subjected to SDS-PAGE (Bio-Rad) and subsequently transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Copenhagen, Denmark). Blots were probed with polyclonal goat anti-human plasminogen (Abcam, Cambridge, MA), polyclonal rabbit anti-human uPA (Abcam), or polyclonal rabbit anti-actin (Abcam). Primary antibodies were detected with horseradish peroxidase (HRP)-coupled antibodies (Dako, Glostrup, Denmark) and ECL system (Amersham Biosciences).
Cloning and siRNA Experiments
Full-length ENaC subunit cDNA was amplified by PCR using specific primers (Supplemental Table 4) and cloned into pcDNA6.2 (Invitrogen). The hexahistidine tag was inserted between residues 148 and 153 (mouse ENaC numbering) by using PCR. All constructs were verified by sequencing (MWG Biotech, Martinsreid, Germany). Subconfluent M-1 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Transfection efficiency was 70 to 75% (Supplemental Figure 6). For NTA-Atto550 (Sigma) labeling, cells expressing the hexahistidine-tagged ENaC subunit were seeded onto coverslips and incubated at 37°C/5% CO2 in culture medium. Twenty four hours after seeding, cells were stimulated with or without plasmin or urine for 5 min, followed by incubation with 10 µM NTA-550 dissolved in DMEM:F12 for 1 min. Cells were washed once with DMEM:F12 and fixed with 4% formaldehyde/PBS. M-1 cells were transfected with siRNA using DharmaFECT1 (Dharmacon, Herlev, Denmark) according to the manufacturer's protocol. The ENaC subunit was knocked down using 75 nM of sequence specific siRNA (siGENOME, cat. no. D-043105-02 and cat. no. D-043105-04; Dharmacon). As a negative control, cells were transfected with Silencer Negative Control (cat. no. AM4611; Ambion, Naerum, Denmark). Knockdown was assessed by reverse transcriptase-quantitative PCR 24 h after transfection, at the protein level by immunofluorescence or functionally after a minimum of 2 d. M-1 cells used for immunofluorescence were transfected with siRNA and seeded onto coverslips and incubated for 48 h at 37°C/5% CO2 in culture medium. After fixation in 4% formaldehyde/PBS, the M-1 cells were incubated with rabbit ENaC antibody (1:200; Sigma) diluted in PBS and 0.5% Tween-20. Subsequently, cells were washed and incubated with Alexa-488 goat anti-rabbit antibody (1:200; Molecular Probes, Tåstrup, Denmark). Cells were counterstained with DAPI, and fluorescence was visualized using appropriate filters.
Immunohistochemistry
Human kidney samples were obtained from randomly selected patients who underwent unilateral nephrectomy for renal cancer at the Department of Urology, Odense University Hospital. All patients gave informed written consent to the use of tissue from the extirpated kidney. The use of tissue was approved by the public regional ethics Committee (File 20010035). Human nephrectomy specimens and cryosections of rat kidneys were fixed in acetone and immunostained with polyclonal rabbit uPA antibody (Abcam) followed by HRP-coupled antibody (Dako). Sections were treated with 0.01% diaminobenzidine (Dako) and 0.02% H2O2 and counterstained with hematoxylin.
Isolation of Oocytes and Two-Electrode Voltage-Clamp Experiments
Isolation of Xenopus laevis oocytes, injection of cRNA, and two-electrode voltage-clamp experiments were performed essentially as described previously.47,48 Details are given in the online supplemental materials.
Detection of ENaC Cleavage Products at the Cell Surface
Biotinylation experiments were essentially performed as described by Harris et al.16 using 30 oocytes per group. All biotinylation steps were performed at 4°C. Oocytes were incubated in the biotinylation buffer containing 10 mM triethanolamine (pH 9.5), 150 mM NaCl, 2 mM CaCl2, and 1 mg/ml EZ-link sulfo-NHS-SS-Biotin (Pierce, Rockford, IL) for 15 min with gentle agitation. The biotinylation reaction was stopped by washing the oocytes twice with quench buffer containing 192 mM glycine and 25 mM Tris-Cl (pH 7.5). Oocytes were then washed three times with ND96 solution and lysed by passing them five times through a 27-G needle in lysis buffer containing 1% Triton X-100 and 1% Igepal CA-630 (Sigma), 500 mM NaCl, 5 mM EDTA, and 50 mM Tris-Cl (pH 7.4), supplemented by a protease inhibitor cocktail (Complete Mini EDTA-Free protease inhibitor cocktail tablets; Roche Diagnostics, Mannheim, Germany). The lysates were incubated for 20 min on ice and centrifuged for 10 min at approximately 13,500 x g. Supernatants were transferred to 1.5-ml Eppendorf tubes containing 100 µl of Immunopure immobilized Neutravidin beads (Pierce) equilibrated with lysis buffer. After overnight incubation at 4°C with gentle agitation, the beads were pelleted for 3 min at approximately 13,500 x g. Supernatants were removed, and beads were washed three times with lysis buffer. A total of 100 µl of 2x SDS-PAGE sample buffer (Rotiload 1; Roth, Karlsruhe, Germany) was added to the beads. Samples were boiled for 5 min at 95°C before loading them on the 10% SDS-PAGE. Monoclonal anti-V5 antibody was obtained from Invitrogen (Karlsruhe) and used at a dilution of 1:5000. HRP-labeled secondary sheep anti-mouse antibodies were purchased from Sigma (Taufkirchen, Germany) and used at a dilution of 1:10,000.
Human Urine Samples and Patient Data
Four healthy control subjects and five patients with nephrotic syndrome were enrolled in the study. The study was approved by the public regional ethics committee (file VF 20040231), and oral and written informed consents were obtained from each participant before the study. The urine was aspirated from 24-h urine samples. All analytical procedures were as described for rat urine.
Statistical Analysis
Data are presented as means ± SEM. Data were compared using the unpaired t test, and variance was tested by the F test and Welch correction was used when appropriate. Multiple comparisons were analyzed by ANOVA and post hoc analysis by Dunnett or Bonferroni multiple comparison test. The level of significance was P < 0.05.
|
NOTE ADDED IN PROOF |
|---|
|
TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSNOTE ADDED IN PROOFDISCLOSURESREFERENCES |
|---|
|
DISCLOSURES |
|---|
|
TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSNOTE ADDED IN PROOFDISCLOSURESREFERENCES |
|---|
|
Acknowledgments |
|---|
The seminal observation that nephrotic urine activates ENaC function was reported in abstract form at the annual meeting of the American Society of Nephrology; October 29 through November 1, 2004; St. Louis, MO (J Am Soc Nephrol 15: 2004, 306A; poster no. SA-PO029).
We thank Mette Svendsen and Dina Dræby for assistance with animal procedures and Inge Andersen, Gitte Dybmose, Mette Fredenslund, Bodil Kristensen, Anette Rasmussen, Lis Teusch, Jessica Ott, and Ralf Rinke for skillful technical assistance. In addition, we thank Søren Andersen for skilled technical assistance with MALDI-TOF mass spectrometry. We thank Anthony M. Carter for linguistic advice.
|
Footnotes |
|---|
P.S. and C.B. contributed equally to this work.
Supplemental information for this article is available online at http://www.jasn.org/.
See related editorial, "Plasmin and Sodium Retention in Nephrotic Syndrome," on pages 233–234.
|
REFERENCES |
|---|
|
TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSNOTE ADDED IN PROOFDISCLOSURESREFERENCES |
|---|
Related Articles
This article has been cited by other articles:
 |
|
 |
|
  J. C. Hu, A. Bengrine, A. Lis, and M. S. Awayda Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage J. Biol. Chem., December 25, 2009; 284(52): 36334 - 36345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Svenningsen, T. R. Uhrenholt, Y. Palarasah, K. Skjodt, B. L. Jensen, and O. Skott Prostasin-dependent activation of epithelial Na+ channels by low plasmin concentrations Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2009; 297(6): R1733 - R1741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Frindt and L. G. Palmer Surface expression of sodium channels and transporters in rat kidney: effects of dietary sodium Am J Physiol Renal Physiol, November 1, 2009; 297(5): F1249 - F1255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Haerteis, B. Krueger, C. Korbmacher, and R. Rauh The {delta}-Subunit of the Epithelial Sodium Channel (ENaC) Enhances Channel Activity and Alters Proteolytic ENaC Activation J. Biol. Chem., October 16, 2009; 284(42): 29024 - 29040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Kleyman, M. D. Carattino, and R. P. Hughey ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases J. Biol. Chem., July 31, 2009; 284(31): 20447 - 20451. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. R. Kleyman and R. P. Hughey Plasmin and Sodium Retention in Nephrotic Syndrome J. Am. Soc. Nephrol., February 1, 2009; 20(2): 233 - 234. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
), whereas the ability of the precipitate to stimulate sodium current and serine protease activity is increased (
). *P < 0.05 (n = 4). (B) Chromatogram of ion exchange chromatography of the aprotinin-affinity precipitate and corresponding serine protease activities (solid line, A280nm; dotted line, conductance of the elution buffer; 
) were used for internal calibration. (D) Knockdown of 
and 
). **P < 0.01, ***P < 0.001.
