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Am J Physiol Renal Physiol 295: F1052-F1062, 2008. First published July 23, 2008; doi:10.1152/ajprenal.00031.2008
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Trypsin can activate the epithelial sodium channel (ENaC) in microdissected mouse distal nephron

Viatcheslav Nesterov,1,* Anke Dahlmann,2,* Marko Bertog,1 and Christoph Korbmacher1

1Institut für Zelluläre und Molekulare Physiologie and 2Medizinische Klinik 4—Nephrologie und Hypertensiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

Submitted 22 January 2008 ; accepted in final form 21 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Proteases are involved in the processing and activation of the epithelial sodium channel (ENaC). The aim of the present study was to investigate whether the prototypical serine protease trypsin can activate ENaC in microdissected, split-open mouse renal distal tubules. Whole-cell patch-clamp recordings from principal cells of connecting tubules (CNT) or cortical collecting ducts (CCD) demonstrated that addition of trypsin (20 µg/ml) to the bath solution increased the ENaC-mediated amiloride-sensitive whole cell current ({Delta}IAmi) in the majority of cells. In contrast, trypsin applied in the presence of an excess of soybean trypsin inhibitor had no stimulatory effect. The {Delta}IAmi response to trypsin was variable, ranging from no apparent effect to a twofold increase in {Delta}IAmi with an average stimulatory effect of 31 or 37% in mice on low-Na+ or standard Na+ diet, respectively. In cultured M-1 mouse collecting duct cells, a robust stimulatory effect of trypsin on {Delta}IAmi was only observed in cells pretreated with protease inhibitors. This suggests that endogenous proteases contribute to ENaC activation in renal tubular cells and that the degree of ENaC prestimulation by endogenous proteases determines the magnitude of the stimulatory response to exogenous trypsin. In conclusion, we provide electrophysiological evidence that trypsin can stimulate ENaC activity in native renal mouse tubules. Thus, in the kidney, ENaC stimulation by extracellular proteases may be a relevant regulatory mechanism in vivo.

microdissected renal tubules; whole-cell patch-clamp recording; serine proteases; epithelial sodium transport; amiloride

THE AMILORIDE-SENSITIVE epithelial sodium channel (ENaC) is the rate-limiting step for sodium absorption in a number of epithelial tissues, including the aldosterone-sensitive distal nephron (ASDN). The latter comprises the late distal convoluted tubule, the connecting tubule, and the entire collecting duct. In the ASDN, the fine-tuning of renal sodium absorption takes place. In this context, the appropriate regulation of ENaC activity in the ASDN is critically important for the maintenance of body sodium balance and, hence, for the long-term regulation of arterial blood pressure. This is evidenced by gain-of-function mutations in ENaC that cause a severe form of salt-sensitive arterial hypertension known as Liddle's syndrome. In contrast, loss-of-function mutations in ENaC cause pseudohypoaldosteronism type 1, characterized by severe renal salt wasting (21, 28, 46).

ENaC is composed of three homologous subunits ({alpha}, β, and {gamma}). The recently published crystal structure of the related acid-sensing ion channel ASIC1 suggests that ENaC is a heterotrimer (27). Each subunit has two transmembrane domains, a large extracellular loop, and cytosolic NH2 and COOH termini. There is recent evidence that proteases contribute to ENaC regulation by cleaving specific sites in the extracellular loops of the {alpha}- and {gamma}-subunits but not the β-subunit (29, 43, 45). However, the physiological relevance of this regulatory pathway and the relevant proteases involved are not yet known.

Western blot analysis revealed the presence of distinct ENaC cleavage products of the {alpha}- and {gamma}-subunits in ENaC expressing cell systems and in native renal tissue (29, 45). In the kidney, the appearance of cleaved ENaC products is enhanced in rats with increased plasma aldosterone levels (34, 35). Importantly, the appearance of cleaved products correlated with increasing ENaC currents (15). Thus the channel is thought to be in its mature and active form in its cleaved state. There is evidence for the presence of both cleaved and noncleaved channels in the plasma membrane (26). This suggests that, at the cell surface, a noncleaved population of ENaC is accessible for proteases to cleave and activate the channel from the extracellular side.

Membrane-anchored channel-activating proteases (CAPs) with extracellular proteolytic activity have been identified using the Xenopus laevis oocyte expression system and were shown to activate ENaC when coexpressed with the channel (54, 55, 57). Various membrane-bound and/or secreted proteases such as CAPs are likely to exist in ENaC-expressing epithelia. One possible candidate for an endogenous ENaC-activating protease in the kidney is prostasin, the mammalian homolog of Xenopus CAP1 (58). Prostasin is an attractive candidate, since its expression is regulated by aldosterone (38), the main hormonal regulator of ENaC. Moreover, prostasin is expressed in the kidney and in cultured collecting duct cells (1, 40). Another candidate is tissue kallikrein (TK), which recently was reported to be involved in ENaC processing in the kidney. TK is synthesized in large amounts in the connecting tubule (CNT) and is released into the urine, from where it could act on ENaC either directly or indirectly by activating other proteases (42). It is likely that additional tissue-specific proteases are involved in ENaC regulation and that channel-activating proteases may be part of a complex protease cascade (45). Finally, endogenous protease inhibitors may add to the complexity of this regulatory pathway (59).

The proteases discussed in the preceding paragraph are thought to cleave and activate the channel from the extracellular side after the channel has been inserted into the plasma membrane. In addition, proteolytic cleavage by furin or related cellular convertase, is thought to be important for ENaC maturation in the biosynthetic pathway before the channel reaches the plasma membrane (25, 52). At present, the relative importance of intra- vs. extracellular proteolytic processing of ENaC is unclear. It is conceivable that, under some conditions, proteolytic processing and channel activation are complete by the time the channel reaches the plasma membrane. On the other hand, partially cleaved channels may reach the plasma membrane, where they await further proteolytic processing and activation.

The mechanism by which channel cleavage results in channel activation also is not yet fully understood. Cleavage-induced release of inhibitory domains from the extracellular loops of the {alpha}- and {gamma}-subunits is thought to be essential for ENaC activation (7, 10), whereas there is no evidence for cleavage of the β-subunit. Proteolytic cleavage of the channel may cause a conformational change and lead to the activation of a population of near-silent channels in the membrane (8, 9). Relieving sodium self-inhibition also may contribute to ENaC activation by proteases (5, 47). At the cell surface, activation of ENaC by extracellular proteases appears to require the cleavage of the {gamma}-subunit, possibly at more than one cleavage site (2, 7, 13, 23). Finally, indirect mechanisms may contribute to ENaC activation by extracellular trypsin (3).

Most of our knowledge about ENaC activation by extracellular proteases stems from insightful studies in model systems such as X. laevis oocytes and cultured cells, including renal epithelial cell lines. Recently, some functional evidence is emerging that ENaC activation by extracellular proteases can indeed occur in native renal tissue (17). In microperfusion studies, it was observed that trypsin treatment enhanced net sodium absorption in isolated rabbit cortical collecting ducts (CCD), at least at slow tubular fluid perfusion rates (36). Moreover, it has been reported that exposure to luminal TK increases the intracellular sodium concentration ([Na+]i) in principal cells of isolated and microperfused mouse CCD. Since the TK-dependent increase in [Na+]i was prevented by amiloride, it was concluded that it is mediated by an activation of ENaC (42). The aim of the present study was to seek electrophysiological evidence for ENaC activation by trypsin in native renal tissue ex vivo. Trypsin was chosen as a prototypical serine protease known to activate ENaC in model systems from the extracellular side. We used conventional whole-cell patch-clamp recordings to test the effect of trypsin on the amiloride-sensitive ENaC whole-cell current in microdissected, split-open mouse tubules.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. For this study we used 6- to 8-wk-old male C57/BL6 mice. It is well known that animals have to be kept on a low-Na+ diet to measure sizeable ENaC whole-cell currents in microdissected tubules from rat and mouse (12, 20). Therefore, animals were routinely maintained on a low-Na+ diet (Na+ content 0.13 g/kg; Altromin, Lage, Germany) with free access to tap water for 2–3 wk before the experiment. Additional experiments also were performed using animals maintained on a corresponding standard Na+ diet (Na+ content 3.2 g/kg).

Preparation of renal tubules. After anesthesia (thiopental sodium, 50 mg/kg ip), the circulatory system was perfused via the left ventricle with Leibovitz medium (LM) containing collagenase (1 mg/ml), soybean trypsin inhibitor (SBTI; 2 µg/ml), aprotinin (2 µg/ml), and amiloride (10 µM). After perfusion, both kidneys were removed, cut into coronal slices, and incubated for 10–15 min at 37°C in the same solution used for the perfusion. After collagenase washout, slices were kept in ice-cold LM containing amiloride (10 µM) and SBTI (2 µg/ml) throughout the microdissection procedure. Cortical tubules were separated manually using fine forceps. We identified and isolated tubular segments with characteristic branching indicative of CNTs and/or CCDs (Fig. 1A). The isolated tubular segments were attached to glass coverslips coated with Cell-Tak (Collaborative Research, Bedford, MA) and transferred to a temperature-controlled perfusion chamber (37°C) mounted on an inverted microscope (Leica DM IRB). To gain access to the apical cell membrane with the patch pipette, we cut tubules open with a broken glass pipette attached to a micromanipulator (Fig. 1B). Principal cells expressing ENaC were identified according to their characteristic shape and their response to amiloride (see below).


Figure 1
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  		Fig. 1. Micrographs of microdissected mouse renal tubules as used for patch-clamp experiments. A: differential interference contrast micrograph of an isolated tubular fragment from the renal cortex as typically used for whole-cell patch-clamp experiments. The fragment was identified by its branching, a characteristic feature of connecting tubules (CNT) and cortical collecting ducts (CCD). B: cut open tubule with access to the luminal side of principal cells. Note on the left the contour of a patch pipette approaching the apical surface of a cell.

 
Cell culture. The M-1 cell line (ATCC 2038CRL; American Type Culture Collection, Rockville, MD) was originally obtained from Dr. G. Fejes-Toth (49). Cells were used from passages 29 to 34 and were handled essentially as described previously (30, 31). Cells were maintained in a 5% CO2 atmosphere at 37°C in PC1 serum-free culture medium (Lonza, Walkersville, MD) supplemented with 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. For the patch-clamp experiments, cells were seeded onto small pieces of glass coverslips, grown to confluence, and used on days 5–7 after seeding when "dome" formation was evident as a sign of active transepithelial fluid and electrolyte transport (30). To inhibit endogenous proteases, we pretreated some batches of M-1 cells overnight with aprotinin (Sigma-Aldrich; 30 µg/ml) and in addition exposed them to a convertase inhibitor for 3–6 h before the patch-clamp experiments. As a convertase inhibitor, we used decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk) from Calbiochem/Merck Biosciences (Schwalbach, Germany). A 10 mM stock solution of dec-RVKR-cmk was prepared in DMSO and was diluted in bath solution to a final concentration of 40 µM (50).

Experimental solutions and chemicals. For the patch-clamp experiments, the pipette solution contained (in mM) 95 K-gluconate, 5 Na-gluconate, 2 Mg-ATP, 10 HEPES, 2 EGTA-Na, 0.2 MgCl2, 40 CsOH, and 20 TEA-OH, with pH adjusted to 7.2 using gluconic acid. Our standard bath solution contained (in mM) 140 Na-gluconate, 5 K-gluconate, 1 CaCl2, 5 barium acetate, 1 MgCl2, 10 HEPES, and 5 glucose, with pH adjusted to 7.4 using Tris. Unless trypsin was added to the bath solution, all bath solutions in experiments with microdissected tubules contained SBTI (20 µg/ml) and/or aprotinin (2 µg/ml) to reduce the risk of a contamination with trypsin or with cellular proteases released in the process of opening the tubules. In experiments with M-1 cells, aprotinin (2 µg/ml) was routinely present in the bath solution unless trypsin was added. Amiloride hydrochloride, collagenase (clostridiopeptidase A type V), trypsin type I from bovine pancreas, aprotinin, and SBTI were purchased from Sigma-Aldrich (Taufkirchen, Germany). Amiloride was prepared as a 10 mM aqueous stock solution.

Electrophysiology. A computer-controlled EPC-9 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany) was used to obtain conventional whole-cell patch-clamp recordings (22) from principal cells in microdissected tubules or from cultured M-1 cells using an experimental protocol and setup essentially as described previously (13, 31, 32). Only recordings with a clear amiloride response were included in our experiments. Pipettes were made from borosilicate glass (GC150-15; Harvard Apparatus) with a resistance of ~5–10 M{Omega}. Seals were formed at the apical cell surface by using gentle suction. The whole-cell configuration was achieved by increasing the suction after the seal resistance had exceeded at least 1 G{Omega}. Series resistance was in the order of 10–30 M{Omega} and was not compensated. With a 3 M KCl flowing boundary electrode, the liquid junction potential occurring at the pipette/bath junction was measured to be ~6 mV. To correct for this, we shifted the current-voltage (I-V) plots to the left by 6 mV. For the whole-cell configuration, the cytoplasmic potential corresponds to the pipette holding potential (Vhold). For continuous whole-cell current recordings, cells were voltage-clamped using a Vhold of –60 mV. Under our experimental conditions, this favors the detection of Na+ inward currents. Inward currents (downward current deflections) are defined as negative currents, i.e., movement of positive charge from the extracellular side to the cytoplasmic side. Starting from a Vhold of –60 mV, voltage step protocols were performed intermittently using consecutive 600-ms step changes to potentials ranging from –100 up to 20 mV in 20-mV increments. The average current values reached during the last 100 ms of the voltage steps were used for the I-V plots. The amiloride-sensitive whole-cell current ({Delta}IAmi) was determined by subtracting the whole-cell current measured in the presence of amiloride from the corresponding current measured in the absence of amiloride. Data were recorded with a sampling rate of 250 Hz and filtered at 100 Hz for display and analysis.

Statistics. Values are means ± SE. Statistical significance was evaluated using the paired Student's t-test, ratio t-test, or Fischer ANOVA test, as appropriate; P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
{Delta}IAmi can be detected in principal cells of microdissected mouse distal tubules under baseline conditions. For our patch-clamp experiments, we used microdissected tubular fragments from the renal cortex, which were identified by their branching, a characteristic feature of CNT and CCD (Fig. 1A). After transfer of the tubular fragments to the perfusion chamber and the opening of the tubules, it was not possible for us to distinguish with certainty whether the patch-clamp experiments were performed on CNT or CCD. However, both nephron segments are known to express ENaC (19). Therefore, the results of all successful experiments with a measurable amiloride-sensitive whole-cell current were pooled and are reported together. Experiments were routinely started in a bath solution containing 10 µM amiloride to inhibit ENaC and to reduce channel rundown (56). For our initial set of experiments, animals were kept on a low-Na+ diet to achieve sizeable ENaC whole-cell currents (12, 20). In the presence of amiloride, the initial {Delta}IAmi at a holding potential of –60 mV averaged –7.8 ± 5.4 pA (n = 26) within the first few minutes after the whole-cell configuration was achieved. The appearance of a sizeable inward current component upon washout of amiloride and the rapid return of the whole-cell current toward its initial level upon readdition of amiloride were taken as evidence that the cell under investigation was a principal cell expressing ENaC. In 26 successful whole-cell recordings from principal cells in microdissected tubules, the initial {Delta}IAmi averaged 194 ± 49 pA at a holding potential of –60 mV. In 23 of these experiments, {Delta}IAmi values could be measured at –60 and –80 mV and were used to calculate the slope conductance between these two points. This amiloride-sensitive whole cell slope conductance averaged 3.0 ± 0.7 nS. The current and conductance values are well within the range of what has been reported for the rat (19) and mouse CCD (12) from animals maintained on a low-Na+ diet.

Trypsin can stimulate {Delta}IAmi in principal cells of microdissected distal tubules from mice maintained on a low-Na+ diet. In Fig. 2, representative whole-cell current traces are shown in which gigaohm seals could be maintained long enough to perform several solution exchanges and to apply drugs while continuously monitoring whole-cell currents. The top trace represents a control experiment in which the bath solution was repeatedly alternated between a solution containing amiloride (10 µM) and a solution without amiloride. It can be seen that each washout of amiloride revealed an inward current component that was rapidly inhibited upon reapplication of amiloride. In control experiments, the magnitude of {Delta}IAmi gradually declined over time, which is a commonly observed phenomenon called channel rundown (56). Under our experimental conditions, we also have to consider the possibility that the protease inhibitors routinely present in the bath solution may contribute to this phenomenon. The middle trace in Fig. 2 shows an experiment in which trypsin (20 µg/ml) was added to the bath solution after two initial amiloride washout maneuvers under control conditions. Interestingly, after the tubule was exposed to trypsin, {Delta}IAmi increased about twofold compared with its initial value. In contrast, trypsin (20 µg/ml) applied in the presence of an excess of SBTI (200 µg/ml) failed to have a stimulatory effect on {Delta}IAmi (bottom trace of Fig. 2). This indicates that the stimulatory effect of trypsin on {Delta}IAmi requires the proteolytic activity of trypsin and is not an artifact of the bath solution exchange.


Figure 2
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  		Fig. 2. Exposure to trypsin increases the amiloride-sensitive whole-cell current ({Delta}IAmi) in microdissected renal tubules of mice maintained on a low-Na+ diet. Shown are 3 representative whole-cell current recordings that were obtained from 3 different microdissected tubules. Continuous whole-cell current recordings were performed at a holding potential of –60 mV. Amiloride (10 µM) was present in the bath solution during the time periods indicated by horizontal bars. Unless trypsin was added, all bath solutions contained 20 µg/ml soybean trypsin inhibitor (SBTI) and/or aprotinin (2 µg/ml) to reduce the risk of a contamination with trypsin or with cellular proteases released in the process of opening the tubules. Current responses resulting from voltage-step protocols performed before each solution exchange were truncated for clarity. Top trace: control experiment that demonstrates a gradual rundown of {Delta}IAmi over time. Middle trace: trypsin (20 µg/ml) was present in the bath solution for the time period indicated and resulted in an almost 2-fold increase in {Delta}IAmi. Bottom trace: trypsin applied in the presence of an excess of 200 µg/ml SBTI had no stimulatory effect on {Delta}IAmi.

 
The response to trypsin is variable. In Fig. 3, results from experiments similar to those shown in Fig. 2 are summarized. Figure 3A demonstrates that the spontaneous rundown of {Delta}IAmi in untreated control cells (solid lines) was similar to that observed in experiments with trypsin application in the presence of an excess of SBTI (dotted lines). Therefore, these data were pooled and used as average control data (Fig. 3C). In contrast to the spontaneous rundown of {Delta}IAmi in control experiments, application of trypsin (in the absence of SBTI) markedly increased {Delta}IAmi in more than half of the experiments (Fig. 3B). Interestingly, the response to trypsin was rather variable, ranging from no apparent effect to a twofold increase in {Delta}IAmi. Figure 3C shows the average time course of {Delta}IAmi from all experiments in which trypsin was applied and compares this to the time course of {Delta}IAmi from all control experiments, which includes untreated time-matched control cells and experiments in which trypsin was applied in the presence of an excess of SBTI (see above). On average, trypsin significantly increased {Delta}IAmi by 31 ± 10% (n = 14; P < 0.05) within 2 min of trypsin application (Fig. 3C).


Figure 3
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  		Fig. 3. Average trypsin effect on {Delta}IAmi in microdissected tubules of mice maintained on a low-Na+ diet. Shown are summary data from experiments similar to those shown in Fig. 2. For each experiment, {Delta}IAmi values were normalized to the value of {Delta}IAmi measured before the application of trypsin or, in control experiments, to the value of {Delta}IAmi measured at the corresponding time point. A: time course of {Delta}IAmi from individual control experiments performed in the absence of trypsin (solid lines) and from control experiments in which trypsin was applied in the presence of an excess of SBTI (dotted lines). B: time course of {Delta}IAmi from individual experiments in which trypsin (20 µg/ml) was applied. C: average data from the experiments shown in A (bullet, dotted line; n = 9) and B ({blacksquare}, solid line; n = 14). On average, exposure to trypsin caused a significant increase in {Delta}IAmi of ~30% ({dagger}P < 0.05). In cells treated with trypsin, the average normalized {Delta}IAmi value remained elevated compared with that of time-matched control cells (*P < 0.05; **P < 0.01).

 
The stimulatory effect of trypsin on {Delta}IAmi is not associated with a loss of sodium selectivity. Figure 4A depicts representative whole cell current traces from an experiment similar to that shown in the middle trace of Fig. 2. The whole-cell current traces were recorded during voltage step protocols that were performed in the presence and absence of amiloride before and after the application of trypsin. Current traces like those shown in Fig. 4A were used to construct average I-V plots of {Delta}IAmi from experiments in which the stimulatory effect of trypsin on {Delta}IAmi was at least 30%. The resulting average I-V plots are shown in Fig. 4B. They suggest that the reversal potential of {Delta}IAmi before and after trypsin application is close to the predicted reversal potential for Na+ (ENa). This suggests that the stimulatory effect of trypsin is not caused by the activation of a nonselective cation conductance. Thus trypsin stimulates a sodium-selective and amiloride-sensitive conductance. This indicates that its stimulatory effect is mediated by an activation of ENaC. It should be pointed out that the incomplete I-V plots shown in Fig. 4B do not permit an accurate determination of the reversal potential of {Delta}IAmi before and after trypsin application. Therefore, we cannot rule out a minor effect of trypsin on the ion selectivity of ENaC.


Figure 4
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  		Fig. 4. Exposure to trypsin has no major effect on the sodium selectivity of the amiloride-sensitive whole-cell current in microdissected mouse tubules. A: typical whole-cell current responses resulting from voltage-step protocols performed before and after exposure to trypsin in the absence and presence of amiloride. Voltage-step protocols were repeatedly performed in an experiment similar to that shown in the middle trace of Fig. 2. Starting from a holding potential of –60 mV, consecutive 600-ms step changes were performed to potentials ranging from –100 up to 20 mV in 20-mV increments. Voltage-step protocols were performed in the absence of amiloride (top traces; Ami–) and in the presence of 10 µM amiloride (middle traces; Ami+) before and after exposure of the cell to 20 µg/ml trypsin (left and right traces, respectively). The bottom traces ({Delta}IAmi) were obtained by subtracting the whole-cell current traces recorded in the presence of amiloride from the corresponding current traces recorded in its absence. B: whole-cell current responses from voltage-step protocols as shown in A were used to obtain average current-voltage (I-V) plots of {Delta}IAmi before (bullet, dotted line) and after exposure to trypsin ({blacksquare}, solid line). The average I-V plots summarize experiments in which trypsin increased {Delta}IAmi by at least 30% (n = 7). The predicted reversal potential for Na+ (ENa) is indicated by an arrow.

 
There is no clear dependency of the trypsin effect on the initial magnitude of {Delta}IAmi. We noticed a high cell-to-cell variability of the initial {Delta}IAmi that ranged from ~50 to 750 pA. In the oocyte expression system, it was previously reported that the magnitude of the stimulatory effect of trypsin on ENaC is inversely correlated with the initial {Delta}IAmi value. This suggested that the trypsin effect was larger in cells expressing a low number of channels or channels with a low open probability before trypsin application (11). To analyze our data in microdissected tubules in this context, we plotted the relative stimulatory effect of trypsin against the initial {Delta}IAmi value (Fig. 5). Interestingly, trypsin failed to stimulate {Delta}IAmi in the two cells with the highest initial {Delta}IAmi, which would be consistent with the concept of an inverse correlation between the trypsin effect and the initial {Delta}IAmi. In cells with an initial {Delta}IAmi ranging from ~50 to 200 pA, trypsin increased {Delta}IAmi in 7 of 12 cells by >30%. In this latter group, there was no apparent correlation among the initial {Delta}IAmi, the responsiveness to trypsin, and the magnitude of the stimulatory effect. Thus, with our limited number of observations, we were unable to confirm a clear dependency of the trypsin effect on the initial {Delta}IAmi value in microdissected tubules.


Figure 5
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  		Fig. 5. Effect of trypsin on {Delta}IAmi in microdissected tubules: relationship to the initial {Delta}IAmi value. For the individual experiments shown in Fig. 3B, the effect of trypsin on {Delta}IAmi is expressed as the ratio of {Delta}IAmi measured after and before exposure to trypsin ({Delta}IAmi-trypsin/{Delta}IAmi-initial) and is reported as a function of the initial {Delta}IAmi value ({Delta}IAmi-initial). Encircled data points represent cells in which trypsin increased {Delta}IAmi by at least 30%.

 
Trypsin also can stimulate {Delta}IAmi in principal cells of microdissected distal tubules from mice maintained on a standard Na+ diet. With the diets used, plasma aldosterone levels are increased to ~1.5 nM in mice maintained on a low-Na+ diet compared with 0.6 nM in mice maintained on standard Na+ diet (4). To investigate whether this difference in the level of plasma aldosterone influences the response to trypsin, we also performed whole-cell current recordings in microdissected tubules from animals maintained on a standard Na+ diet. In this set of experiments we detected typical amiloride-sensitive whole-cell currents (Fig. 6A) in 21 of 33 successful whole-cell recordings. At a holding potential of –60 mV, {Delta}IAmi averaged 58 ± 13 pA (n = 21). Using the current values measured at –60 and –80 mV, we estimated an average amiloride-sensitive whole-cell slope conductance of 1.3 ± 0.3 nS (n = 21), which was significantly lower than that measured in the microdissected tubules of mice maintained on a low-Na+ diet (P < 0.05). However, in tubules from mice maintained on normal Na+ diet, the relative stimulatory effect of trypsin was similar to that observed in tubules of mice on a low-Na+ diet (Fig. 6A). The effect of trypsin was also rather variable, with an average stimulatory effect of 37 ± 16% (n = 12; Fig. 7, A and C). In control experiments, trypsin application in the presence of an excess of SBTI had no significant stimulatory effect on {Delta}IAmi (Figs. 6B and 7, B and C). Interestingly, in one of seven recordings with no measurable {Delta}IAmi at the beginning of the recording, the subsequent application of trypsin resulted in the appearance a of an amiloride-sensitive whole-cell current component of 30 pA. This experiment is not included in the summary data shown in Fig. 7 but is in good agreement with recently published results in microdissected rat CCD (17).


Figure 6
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  		Fig. 6. Exposure to trypsin increases {Delta}IAmi in microdissected renal tubules of mice maintained on a standard Na+ diet. Shown are 2 representative whole-cell current recordings that were obtained from different microdissected tubules. Experiments were performed as described in Fig. 2. Top trace: trypsin (20 µg/ml) was present in the bath solution for the time period indicated. Bottom trace: trypsin was applied in the presence of an excess of SBTI.

 

Figure 7
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  		Fig. 7. Average trypsin effect on {Delta}IAmi in microdissected tubules of mice maintained on a normal Na+ diet. Shown are summary data from experiments similar to those shown in Fig. 6. A: time course of {Delta}IAmi from individual experiments in which tubules were exposed to trypsin (20 µg/ml). B: time course of {Delta}IAmi from individual experiments in which trypsin was applied in the presence of an excess of SBTI. C: average data from the experiments shown in A (bullet, solid line, n = 12) and B ({blacksquare}, dotted line; n = 9). On average, exposure to trypsin caused a significant increase in {Delta}IAmi ({dagger}P < 0.05 vs. initial {Delta}IAmi; *P < 0.05 vs. {Delta}IAmi of time-matched control cells).

 
The responsiveness to trypsin is enhanced in M-1 cells pretreated with protease inhibitors. A variable degree of ENaC preactivation by endogenous cellular proteases, and/or by proteases released during tissue isolation and microdissection, may account for the observed variability of the trypsin effect on {Delta}IAmi in different tubules (see DISCUSSION). We hypothesized that preventing ENaC preactivation by inhibiting endogenous proteases should enhance the stimulatory effect of exogenous trypsin on {Delta}IAmi. Pilot experiments demonstrated that preincubation of microdissected tubules with protease inhibitors for several hours resulted in a loss of viability of the tubules and made it impossible to perform patch-clamp experiments on these tubules. Therefore, we decided to investigate this issue in cultured M-1 mouse CCD cells known to express ENaC when grown to confluence in PC-1 culture medium containing a high concentration (5 µM) of dexamethasone (30–32). As illustrated in the whole-cell current recordings shown in Fig. 8, washout and reapplication of amiloride (2 µM) revealed a sizeable {Delta}IAmi in confluent M-1 cells, confirming functional ENaC expression. In control experiments in M-1 cells without pretreatment, exposure to trypsin had no appreciable stimulatory effect on {Delta}IAmi in the majority of cells (Fig. 8A). Nevertheless, in 5 of 14 experiments, a variable stimulatory effect of trypsin on {Delta}IAmi was observed, which ranged from ~30 to 100%. In summary, {Delta}IAmi averaged 43 ± 4 pA before and 48 ± 7 pA (n = 14) after exposure to trypsin. In the context of an expected spontaneous ENaC rundown (56), these average data suggest a small stimulatory trend that did not reach statistical significance (Fig. 9A). Thus, under control conditions, M-1 cells seem to be less responsive to trypsin than the native tubules. In M-1 cells pretreated with aprotinin overnight, {Delta}IAmi averaged 119 ± 16 pA before and 134 ± 20 pA (n = 20) after exposure to trypsin. Thus, although aprotinin pretreatment enhanced ENaC baseline currents in cultured M-1 cells, possibly by preventing nonspecific toxic effects of proteases released from damaged cells, it did not reveal a clear stimulatory effect of trypsin (Fig. 9B). In contrast, in M-1 cells pretreated for 3–6 h with the convertase inhibitor dec-RVKR-cmk or pretreated with a combination of aprotinin (overnight) plus 3–6 h of convertase inhibitor, application of trypsin caused a pronounced stimulation of {Delta}IAmi in the majority of experiments, ranging from a 25 to 330% increase in {Delta}IAmi. Figure 8B shows a representative whole-cell current recording illustrating the stimulatory effect of trypsin on {Delta}IAmi in M-1 cells pretreated with a combination of aprotinin and the convertase inhibitor dec-RVKR-cmk. On average, trypsin increased {Delta}IAmi from 70 ± 27 to 94 ± 30 pA (n = 10, P < 0.01) in M-1 cells pretreated with the convertase inhibitor alone and from 43 ± 8 to 83 ± 22 pA (n = 11, P < 0.05) in M-1 cells pretreated with both protease inhibitors. Trypsin application caused an average relative increase of {Delta}IAmi by 56 ± 21% (n = 10, P < 0.01) or 111 ± 29% (n = 11, P < 0.05) in M-1 cells pretreated with convertase inhibitor alone or with both protease inhibitors, respectively (Fig. 9, C and D). These data support the hypothesis that the degree of preactivation by endogenous proteases affects the responsiveness of ENaC to exogenous trypsin.


Figure 8
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  		Fig. 8. Representative whole-cell current recordings in cultured M-1 mouse CCD cells. Experiments were performed in confluent M-1 cells grown on glass coverslips at a holding potential of –60 mV using a protocol similar to that for the experiments in microdissected tubules. A: control experiment with application of trypsin to a nontreated M-1 cell. B: application of trypsin to an M-1 cell pretreated with protease inhibitors (aprotinin, 30 µg/ml overnight and convertase inhibitor dec-RVKR-cmk, 40 µM for 3–6 h).

 

Figure 9
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  		Fig. 9. Pretreatment of M-1 cells with protease inhibitors reveals a stimulatory effect of trypsin on {Delta}IAmi. Shown are summary data from experiments similar to those shown in Fig. 8. For each experiment, {Delta}IAmi values were normalized to the value of {Delta}IAmi measured before the application of trypsin. Average time courses are shown for nontreated control cells (A; n = 14) and for M-1 cells pretreated overnight with 30 µg/ml aprotinin (B; n = 20), for 3–6 h with 40 µM convertase inhibitor (C; n = 10), or with a combination of both (D; n = 11). {dagger}P < 0.05 vs. initial {Delta}IAmi; *P < 0.05 vs. {Delta}IAmi of untreated control cells.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Trypsin can activate ENaC in native renal tubules. As far as we know, this is the first study to provide detailed electrophysiological evidence that in native renal tubules, ENaC can be activated by an extracellular serine protease. Using continuous whole-cell patch-clamp recordings, we demonstrated that application of the prototypical serine protease trypsin can stimulate the amiloride-sensitive whole-cell current in principal cells of microdissected, split-open tubular segments from CNT and CCD known to express ENaC. Our results are in good agreement with the previously observed trypsin-induced increase of net sodium absorption in isolated, perfused rabbit tubules (36). In this latter study, net sodium absorption in trypsin-treated CCD significantly exceeded that measured in control tubules by ~70%, which is in a range similar to the stimulatory effect of trypsin observed in the present study. Our findings also are consistent with the recent report that application of TK elicits an amiloride-sensitive increase in [Na+]i in principal cells of isolated and microperfused mouse CCD (42). Together, these studies provide proof of principle that extracellular serine proteases can stimulate ENaC activity in native renal tissue.

Size and variability of trypsin's stimulatory effect on ENaC. In our study, the stimulatory effect of trypsin in native renal tubules was highly variable, ranging from no apparent effect to a twofold increase in {Delta}IAmi, with an average stimulatory effect of ~30%. In the ASDN, this degree of ENaC stimulation would be sufficient to be physiologically relevant for the fine-tuning of renal sodium reabsorption. In the native tubule, the stimulatory effect of trypsin on ENaC whole-cell currents was usually smaller than that typically observed in the X. laevis oocyte expression system. However, the amplitude of the trypsin effect on ENaC also is quite variable in the oocyte expression system, similar to our finding in the native tubule. In oocytes expressing rat ENaC, the trypsin-induced increase in {Delta}IAmi ranged from a hardly detectable increase to a sixfold increase, with an average increase of about threefold (11). In several types of cultured epithelial cells, the stimulatory effect of trypsin on ENaC currents also is smaller than that observed in the oocyte expression system and even may be absent under standard culture conditions. Interestingly, in epithelial cells a stimulatory effect of trypsin on ENaC can be revealed or greatly enhanced by pretreating the cells with aprotinin (6, 14, 33, 37, 44, 53, 54, 58). This is in good agreement with our finding in M-1 cells, in which a stimulatory effect of trypsin was rarely observed under standard culture conditions, whereas the stimulatory effect of trypsin was evident (up to 4-fold) after the M-1 cells were pretreated with protease inhibitors. Interestingly, trypsin had a clear stimulatory effect on {Delta}IAmi in M-1 cells pretreated with the convertase inhibitor, whereas this was not evident in cells pretreated with aprotinin alone. However, the strongest stimulatory effect of trypsin was observed in cells pretreated with both inhibitors, which suggests that the inhibitors act synergistically. Aldosterone has been reported to increase the expression of prostasin at the mRNA and protein level in M-1 cells (38). For our patch-clamp experiments, the M-1 cells were cultured in PC-1 tissue culture medium containing 5 µM dexamethasone (31). At this concentration, dexamethasone is likely to mimic the effect of aldosterone on prostasin expression in the M-1 cells. This may explain the relatively small or absent stimulatory effect of trypsin in M-1 cells under standard culture conditions. Collectively, the findings suggest that the expression level and/or activity of endogenous proteases capable of activating ENaC is relatively high in cultured epithelial cells, whereas it is rather low in oocytes. This latter conclusion also is supported by the finding that heterologous overexpression of mCAP1 stimulates ENaC currents in oocytes and prevents the stimulatory effect of trypsin (58). Thus it is conceivable that the size of the trypsin effect on ENaC in the native tubule will depend on the expression level of endogenous proteases and the degree of proteolytic preactivation of the channel before the application of an extracellular protease. In this context, we have to consider the possibility that the protease inhibitors present in the solution used for kidney perfusion and microdissection and in the bath solution used for superfusing the tubules may have prevented membrane-delimited proteases (e.g., CAPs) from activating ENaC. However, our experiments in M-1 cells suggest that our pretreatment of microdissected tubules with protease inhibitors was not long enough to establish a sizeable inactive pool of channels susceptible to activation by trypsin. An overnight pretreatment of M-1 cells with aprotinin failed to significantly increase the stimulatory response to trypsin, and a 3- to 6-h preincubation of the cells with a membrane-permeable convertase inhibitor (alone or in combination with aprotinin) was needed to achieve a significant effect. Moreover, tissue preparation and microdissection were performed on ice, which is likely to prevent trafficking of noncleaved ENaC to the membrane. Thus it is unlikely that, in the microdissected tubules, the observed stimulatory effect of trypsin depended on the pretreatment with protease inhibitors. Nevertheless, it will be an important task for future research to study the regulation of endogenous renal proteases relevant for ENaC regulation. The activity of endogenous proteases is known to be regulated in a complex manner and is likely to involve signaling pathways and hormones known to affect ENaC activity, such as aldosterone, for example (38). Thus, in animals maintained on a low-Na+ diet, the activity of endogenous proteases may be high enough to preactivate the majority of ENaC present at the plasma membrane. This may explain the lack of a stimulatory effect of trypsin in some of our experiments in animals maintained on a low-Na+ diet and is consistent with the apparent trend of a slightly larger and more robust stimulatory effect of trypsin in animals maintained on a standard Na+ diet. This interpretation is also consistent with results of a recent study published while our manuscript was under review (17). In this study, the authors did not observe a stimulatory effect of trypsin on ENaC currents in microdissected CCD of rats maintained on a Na+-deficient diet. In contrast, in animals maintained on a standard Na+ diet, small but measurable amiloride-sensitive whole-cell currents could be activated by treating the tubules with trypsin. These latter findings are in good agreement with our results obtained in microdissected tubules of mice on a standard Na+ diet. In addition, the authors reported that cleaved ENaC fragments were more abundant in Na+-depleted animals than in those maintained on standard Na+ diet. Thus proteolytic preactivation of ENaC appears to be more pronounced in animals requiring a high ENaC activity to maintain in sodium balance. The reported lack of ENaC stimulation by trypsin in Na+-depleted rats appears to be in contrast to the stimulatory effect observed in the present study in mice maintained on a low-Na+ diet. Possible explanations for this discrepancy are species differences or differences in the experimental conditions. For example, we suspect that the rats used by Frindt et al. (17) may have been more severely Na+-depleted than the mice used in our present study. Indeed, in a previous publication by the same group, it was reported that Na+-depleted rats maintained for 10 days on a very low-Na+ diet containing 3.8 mg/kg Na+ had an average plasma aldosterone level of 28 nM (18). This value is considerably higher than the 1.5 nM observed in mice maintained on a more moderate low-Na+ diet containing 0.13 g/kg Na+ (4), which was the diet used in the present study. In a situation with near-maximal ENaC activation by very high plasma aldosterone levels, an additional stimulation of ENaC by trypsin may be difficult to detect given the high variability of this effect.

Possible reasons for the variability of the trypsin effect on ENaC. In addition to the role of endogenous proteases discussed above, several other mechanisms may contribute to the observed variability of the trypsin effect in microdissected tubules. 1) In rabbit CCD, it has been shown that a trypsin-induced increase in sodium reabsorption rate is abolished at high tubular flow rates (36). In our experiments, we did not systematically vary the flow rate of the bath solution. However, flow rates were not perfectly controlled and monitored. Moreover, the position and orientation of the tubules in the flow chamber and local turbulences may result in nonuniform local flow rates. Thus laminar shear stress acting on the cells under investigation may have been different in different tubular preparations. Therefore, we cannot rule out the possibility that in some experiments a flow-induced activation of ENaC may have occurred and may have obscured a subsequent activation by trypsin, analogous to the findings reported in microperfused rabbit CCD (36).

2) The ENaC-mediated sodium transport rates vary in different parts of the ASDN, with higher {Delta}IAmi values in CNT compared with CCD (19). Thus it is possible that the degree of proteolytic preactivation of ENaC, and hence the ability of extracellular proteases to further stimulate ENaC, may be different in CNT and CCD under certain physiological conditions. Since in our patch-clamp experiments it was impossible for us to distinguish with certainty between principal cells of the CNT or CCD, we cannot rule out the possibility that a different responsiveness of the two nephron segments contributed to the observed variability of the trypsin effect. One may speculate that the highest initial {Delta}IAmi values were measured in segments of the CNT and that in these experiments a further activation of ENaC currents by trypsin was not possible because of the presence of maximally preactivated channels.

3) The relatively moderate and highly variable stimulatory effect of trypsin in tubular cells also may be caused by channel preactivation through locally released cellular proteases from damaged cells during tissue preparation and/or during the microdissection procedure, although we tried to prevent this by including aprotinin and/or SBTI in the solutions.

4) Finally, we have to consider the possibility that the collagenase used for tissue isolation may have contributed to a proteolytic preactivation of ENaC, thereby reducing the subsequent trypsin effect. Therefore, we searched for putative collagenase cleavage sites in the ENaC sequence. No sites corresponding to the common putative collagenase cleavage sites Pro-X-Gly-Pro, Ala-X-Gly-Pro or Pro-X-Gly-X-Pro can be found in the extracellular loops of the three subunits of mouse ENaC. However, the prediction of collagenase cleavage sites is not straight forward (24, 48). Moreover, the collagenase used for the tissue isolation may indirectly affect ENaC through the activation of other proteases. Finally, a contamination of the collagenase preparation with trace amounts of other proteases may have resulted in ENaC preactivation in some of the microdissected tubules. In the context of these possible mechanisms to impede or prevent the detection of a trypsin-induced activation of ENaC in microdissected renal tubules, our finding of a variable trypsin effect is perhaps not surprising.

(Patho-)physiological relevance of ENaC activation by extracellular proteases. The process of ENaC activation by extracellular cleavage may provide a fast mechanism to change the gating properties of channels that are already present in the apical cell membrane. Thus a rapid recruitment of so-called near-silent channels (8, 9) into channels with a higher open probability by extracellular proteases may precede other regulatory mechanisms such as the biosynthesis and insertion of newly synthesized channels. The observed moderate range of ENaC stimulation by trypsin in the native tubule may be sufficient for a fast, initial adaptation of the organism to the need of an increased sodium reabsorption rate. In addition, a regulatory effect of proteases on ENaC also may be involved in the pathogenesis of disease. Studies performed in respiratory epithelial cells suggest that the proteolytic activation of ENaC may aggravate symptoms of cystic fibrosis during acute respiratory infections associated with the generation of local proteases, e.g., neutrophil elastase (8, 9, 23). Similarly, regulation of ENaC activity by proteases may be involved in the pathogenesis of arterial hypertension and in states of sodium retention in the context of renal disease. For example, in patients with crescentic glomerulonephritis, the urinary concentration of neutrophil elastase was found to be significantly higher than in healthy controls (39). Since neutrophil elastase has been shown to activate ENaC in the oocyte expression system (23) and in respiratory epithelial cells (8), it is tempting to speculate that an enhanced cleavage of ENaC by urinary neutrophil elastase may contribute to the development of sodium retention and arterial hypertension associated with acute renal inflammatory diseases. Nephrotic syndrome also is characterized by renal sodium retention, and it has been speculated that this may be caused by ENaC stimulation. It has been reported that in PAN-nephrotic rats, ENaC activation is secondary to hyperaldosteronism (34). However, recent findings suggest that in nephrotic syndrome, filtered plasminogen appears in the urine, where it is converted to plasmin by tubular urokinase-type plasminogen activator and may contribute to ENaC activation (16, 41, 51). This appears to be an attractive mechanism to explain early sodium retention in nephrotic syndrome in the absence of overt hypoalbuminemia and secondary hyperaldosteronism. Renal disease may not only affect the activity of proteases but also may shift the balance between endogenous proteases and protease inhibitors. In this context it is of interest that serpinh1, a serine protease inhibitor, was recently identified in an integrated genomic-transcriptomic approach as a hypertension-related gene (60). These pathophysiological aspects of proteolytic ENaC regulation await further investigation and the identification of relevant proteases. In the future, this may lead to new diagnostic and therapeutic concepts. For example, specific protease inhibitors may become valuable therapeutic tools to prevent excessive ENaC activation under certain pathophysiological conditions.

Conclusion. In conclusion, this study provides electrophysiological evidence that ENaC can be stimulated in native renal tissue by the extracellular application of trypsin. Thus proteases acting on ENaC from the extracellular surface may play a role in renal ENaC regulation in vivo. The identity of these proteases remains to be determined and will be an important area of future research. Moreover, studies are needed to evaluate the role of endogenous proteases in the regulation of ENaC function under physiological and pathophysiological conditions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Deutsche Forschungsgemeinschaft Grant SFB423, TP-A12 (to C. Korbmacher), the Deutsche Nierenstiftung (A. Dahlmann), and an International Society of Nephrology fellowship (to V. Nesterov).


   ACKNOWLEDGMENTS
 
The expert technical assistance of Jessica Ott is gratefully acknowledged.

Present address of V. Nesterov: Novosibirsk Medical University, Krasny Prospect 52, Novosibirsk, Russia.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Korbmacher, Institut für Zelluläre und Molekulare Physiologie, Waldstr. 6, 91054 Erlangen, Germany (e-mail: christoph.korbmacher{at}physiologie2.med.uni-erlangen.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* V. Nesterov and A. Dahlmann contributed equally to this work. Back


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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.
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