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Am J Physiol Renal Physiol 296: F46-F53, 2009. First published October 22, 2008; doi:10.1152/ajprenal.00127.2008
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Delayed and acute effects of interferon-{gamma} on activity of an inwardly rectifying K+ channel in cultured human proximal tubule cells

Kazuyoshi Nakamura, You Komagiri, Toshiyuki Kojo, and Manabu Kubokawa

Department of Physiology, Iwate Medical University School of Medicine, Morioka, Japan

Submitted 12 March 2008 ; accepted in final form 15 October 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The activity of an inwardly rectifying K+ channel in cultured human renal proximal tubule cells (RPTECs) is stimulated and inhibited by nitric oxide (NO) at low and high concentrations, respectively. In this study, we investigated the effects of IFN-{gamma}, one of the cytokines which affect the expression of inducible NO synthase (iNOS), on intracellular NO and channel activity of RPTECs, using RT-PCR, NO imaging, and the cell-attached mode of the patch-clamp technique. Prolonged incubation (24 h) of cells with IFN-{gamma} (20 ng/ml) enhanced iNOS mRNA expression and NO production. In these cells, a NOS inhibitor, N{omega}-nitro-L-arginine methyl ester (L-NAME; 100 µM), elevated channel activity, suggesting that NO production was so high as to suppress the channel. This indicated that IFN-{gamma} would chronically suppress channel activity by enhancing NO production. Acute effects of IFN-{gamma} was also examined in control cells. Simple addition of IFN-{gamma} (20 ng/ml) to the bath acutely stimulated channel activity, which was abolished by inhibitors of IFN-{gamma} receptor-associated Janus-activated kinase [P6 (1 µM) and AG490 (10 µM)]. However, L-NAME did not block the acute effect of IFN-{gamma}. Indeed, IFN-{gamma} did not acutely affect NO production. Moreover, the acute effect was not blocked by inhibition of PKA, PKG, and phosphatidylinositol 3-kinase (PI3K). We conclude that IFN-{gamma} exerted a delayed suppressive effect on K+ channel activity by enhancing iNOS expression and an acute stimulatory effect, which was independent of either NO pathways or phosphorylation processes mediated by PKA, PKG, and PI3K in RPTECs.

nitric oxide; patch-clamp; RT-PCR; DAF

IN THE RENAL PROXIMAL TUBULE cell, basolateral K+ channels play important roles in the formation of cell-negative potential and K+ recycling coupled with Na+-K+-ATPase, both of which provide a driving force for transepithelial Na+ reabsorption (6, 9). An inwardly rectifying K+ channel with an inward conductance of ~40 pS is the most frequently observed K+ channel in cultured human proximal tubule cells under the basal condition (21). Although it is not clear whether the 40-pS K+ channel corresponds to the native basolateral one because of loss of polarity in the cultured cells (20), this K+ channel is an important channel which contributes to the potential formation in human proximal tubule cells (22).

We have previously reported that the activity of the 40-pS K+ channel was modulated at least in part by nitric oxide (NO). The effect of NO on channel activity was suggested to be dose dependently biphasic, since a NO donor, sodium nitroprusside (SNP), stimulated channel activity at 10 µM, but suppressed it at 1 mM (22). Moreover, we demonstrated that inducible NO synthase (iNOS) was present in cultured human proximal tubule cells, suggesting that intracellular iNOS was involved in endogenous NO production in proximal tubule cells (19). The observations that a NOS inhibitor suppressed and a NOS substrate stimulated channel activity (19) also indicate that endogenous NO contributed to the modulation of basal activity of the 40-pS K+ channel.

Proinflammatory cytokines, such as IFN, IL-1, and TNF, are reported to enhance iNOS expression (1). Thus it is possible that they would modulate K+ channel activity by affecting NO production in proximal tubule cells. In fact, some cytokines were reported to affect solute transport in kidney epithelia. For example, TNF-{alpha} and IL-1 inhibited Rb+ uptake in rat thick ascending limb of Henle's loop (5). Furthermore, IFN-{gamma} , IL-1, and TNF-{alpha} were reported to affect the renal transport of Na+ and glucose during severe inflammation (26, 27). However, there are few reports concerning the effects of cytokines on K+ channel activity in renal tubular cells. Until now, only Wei et al. (31) reported that TNF stimulated activity of an apical 70-pS K+ channel in the thick ascending limb of rat kidney (31).

In this study, we investigated the effects of IFN-{gamma} on K+ channel activity in cultured human proximal tubule cells, using RT-PCR, fluorescent NO imaging, and the cell-attached mode of the patch-clamp technique. We demonstrates that IFN-{gamma} exerted a delayed suppressive effect, which was NO dependent, and an acute stimulatory effect, which was NO independent, on K+ channel activity.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Cell culture. Renal proximal tubule epithelial cells (RPTECs) of normal kidney origin (strain 11678, lot 4F0002) were purchased from Cambrex (Walkersville, MD). It is guaranteed that >90% of the cells are positive for {gamma}-GTP, a marker protein specific to the proximal tubule (8). These cells were provided as secondary cultures and maintained up to passage 6 in renal epithelial growth medium (Cambrex) in a humidified atmosphere of 5% CO2-95% air at 37°C. After reaching 70–80% confluence, the RPTECs were used in experiments. In patch-clamp and NO imaging, the cells were dispersed with trypsin/EDTA, resuspended in renal epithelial growth medium, and seeded on collagen-coated coverslips (Asahi Techno Glass, Tokyo, Japan) in {phi}15- or 35-mm dishes at a density of 1 x 105 cells/dish. After 3- to 7-h incubation, the coverslips were transferred to an open bath-heating chamber mounted on an inverted microscope for path-clamp or fluorescent imaging. In RT-PCR analysis, the cells were lysed and total RNA was extracted by using an RNeasy Mini kit (Qiagen, Hilden, Germany).

Solutions. The control bath solution contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES. The pipette solution contained (in mM) 145 KCl, 1 MgCl2, 1 EGTA, and 5 HEPES. These solutions were titrated to pH 7.3 with NaOH or KOH.

Test substances. IFN-{gamma} was purchased from PeproTech EC (London, UK). L-arginine (L-Arg), N{omega}-nitro-L-arginine methyl ester (L-NAME), SNP, a SOD mimetic, tempol, and a phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, were purchased from Sigma (St. Louis, MO). Janus activated kinase (JAK) inhibitors P6 (10) and AG490 (13), a PKA inhibitor, KT5720, a PKG inhibitor, KT5823, and a PI3K inhibitor, LY294002, were from Calbiochem (La Jolla, CA). A fluorescent probe for NO, diaminofluoroscein-2 diacetate (DAF-2DA), was obtained from Daiichi Pure Chemicals (Tokyo, Japan). Wortmannin, P6, AG490, KT5720, KT5823, and DAF-2DA were dissolved in DMSO as stock solutions, whereas the others were dissolved in water. These stock solutions were diluted with the control bath solution before use and added to the bath by hand pipetting. The final concentration of DMSO in the patch bath ranged from 0.039 to 0.054%, which did not affect channel activity.

Patch-clamp technique. Single-channel currents were recorded by cell-attached patches applied to the surface membrane of single RPTECs. All patch-clamp experiments were performed at 33°C. Patch pipettes were fabricated from borosilicate glass capillaries (GC150-7.5, Warner, Hamden, CT), with the resistance ranging from 3 to 4 M{Omega} when filled with the KCl solution. The pipette holding potential (Vp) was set at 0 mV. Current signals were recorded with a patch-clamp amplifier (Axopatch 700B, Molecular Devices, Sunnyvale, CA) and stored on a DAT recorder (RD-120TE, TEAC, Tokyo, Japan). The recorded signals were then low-pass filtered (3611 Multifunction Filter, NF electronic instruments, Tokyo, Japan) at 500 Hz and digitized at a rate of 2.5 kHz through an interface (Digidata 1440A, Molecular Devices). The acquired data were analyzed with pCLAMP10 software (Molecular Devices). Current traces of downward deflections represented inward currents. Channel activity was determined by NPo, which was calculated from an amplitude histogram as

Formula
where N is the maximum number of open channels observed during a given time period in the patch, Po is the open probability, n is the number of channels observed at the same time, and tn is the probability that n channels are simultaneously open. Since the control values of NPo varied among patches, we calculated normalized channel activity (NPo,e/NPo,c) to conveniently compare the channel activity in experimental conditions with controls. NPo,c and NPo,e are the channel activities under control and experimental conditions, respectively. Routinely, we determined NPo,c from a 20-s sampling period just before adding the substance when the steady state lasted for at least 60 s. NPo,e was determined from a 20-s sampling period extracted from the steady state for at least 20–30 s made by the experimental substance.

Reverse transcription and real-time PCR. One microgram of total RNA was reverse transcribed, using an RNA PCR kit (AMV Ver.3.0, TaKaRa Bio, Otsu, Japan). The iNOS mRNA expression were analyzed by quantitative real-time PCR, using the 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA), Power SYBR Green Master Mix (Applied Biosystems), and specific primers (0.4 µM). The primers for human iNOS were 5'-GCCTCGCTCTGGAAAGA-3' (sense) and 5'-TCCATGCAGACAACCTT-3' (antisense), amplifying a 499-bp product (11). The primers for human GAPDH were 5'-GAAGGTGAAGGTCGGAGTC-3' (sense) and 5'-GAAGATGGTGATGGGATTTC-3' (antisense), amplifying a 226-bp product (7). The standard curve for each primer set was generated from serial dilutions (1x, 3x, 10x, 30x, 100x, 300x) of the RPTECs' cDNA. All samples were amplified in duplicate, and the average quantities were used for calculation. The levels of iNOS mRNA expression were calibrated by GAPDH mRNA expression and then compared between control and experimental cells. In each PCR amplification, we also included a sample without reverse transcription in parallel, which served as a negative control. The PCR program was as follows: First, the Taq polymerase was activated by heating it at 95°C for 10 min. This was followed by 45 cycles of the sequential steps consisting of 94°C for 15 s, 55°C for 30 s, and 72°C for 60 s. Electrophoresis of the amplified PCR products was also performed on a 2% agarose gel containing 0.1 µg/ml ethidium bromide, and the PCR products were visualized with UV light of 312 nm. The nucleotide sequences of PCR products were analyzed by TaKaRa Bio and were subjected to BLAST sequence similarity searching.

NO imaging. Production of intracellular NO was evaluated by fluorescent microscopy. RPTECs on glass coverslips were loaded with a membrane-permeant probe, DAF-2DA (10 µM), for 40 min at 37°C. DAF-2DA is hydrolyzed in the cells to the membrane-impermeant DAF-2, which rapidly and irreversibly interacts with NO to generate a highly fluorescent product, DAF-2T. Due to the irreversible interaction between DAF-2 and NO, the intensity of the fluorescent signal represents a cumulative amount of NO produced in the cells, but not the real-time kinetics of NO production. After the probe loading, the cells were thoroughly washed with the control bath solution and placed in a perfusion chamber mounted on a fluorescent microscope. The trypan blue exclusion test indicated that DAF-2 loading did not affect cell viability. The fluorescent signal of DAF-2T was recorded with the InCyt Basic IM imaging system (Intracellular Imaging, Cincinnati, OH) by employing single excitation and emission wavelengths of 470 and 515 nm, respectively. We expressed intracellular NO production as absolute or relative (F/F0) fluorescent intensity of DAF-2T. F represents fluorescent intensity obtained during experiments, and F0 is its basal intensity.

Statistics. Data are expressed as means ± SE. Student's t-test or ANOVA in conjunction with a Bonferroni t-test was used for statistical comparisons. A P value <0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We first examined the effect of IFN-{gamma} on iNOS mRNA expression in RPTECs. The cells were incubated with or without 20 ng/ml IFN-{gamma} for 24 h and subjected to RT-PCR analysis. It is well known that binding of IFN-{gamma} to the type II IFN receptor (IFNGR) activates JAK1 and JAK2 to initiate intracellular signaling pathways which induce various biological actions. Thus a subset of cells was incubated with IFN-{gamma} in the presence of a JAK inhibitor, P6 (1 µM). As shown in Fig. 1A, IFN-{gamma} alone enhanced the intensity of a 499-bp band for iNOS compared with the control. It was also evident that the IFN-{gamma}-induced iNOS expression was suppressed by the concomitant incubation with P6. Quantitative real-time PCR revealed that iNOS mRNA expression was 44-fold higher in IFN-{gamma}-treated cells than in control cells and was only 1.6-fold higher in the cells treated with IFN-{gamma} and P6 (Fig. 1B). We further explored whether the endogenous production of NO actually increased in IFN-{gamma}-treated cells, using fluorescent NO imaging. After 24-h incubation with or without IFN-{gamma}, the cells were loaded with 10 µM DAF-2DA for the evaluation of NO production. With regard to IFN-{gamma}-treated cells, DAF-2 loading was performed in the presence or absence of a NOS inhibitor, L-NAME (100 µM). Representative photographs of NO imaging showed that the fluorescent signals were greater in IFN-{gamma}-treated cells than in control cells, which was abolished by L-NAME (Fig. 1C). The effect of L-NAME strongly suggested that the increased fluorescent signals in IFN-{gamma}-treated cells were due to increased NO production by NOS. The absolute fluorescent intensity, which we quantified in 20 cells, was 1,423.5 ± 56.9 arbitrary units in control cells, 7,296.1 ± 73.9 arbitrary units in IFN-{gamma}-treated/L-NAME (–) cells, and 823.15 ± 52.6 arbitrary units in IFN-{gamma}-treated/L-NAME (+) cells (Fig. 1D). These results indicated that IFN-{gamma} stimulated NO production in RPTECs by enhancing iNOS mRNA expression through activation of JAK.


Figure 1
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  		Fig. 1. Effects of IFN-{gamma} on inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide (NO) production. A: electrophoretic profile of the RT-PCR products for iNOS and GAPDH from renal proximal tubule epithelial cells (RPTECs). M represents the base pair (bp) size marker. C, control cells; I, cells treated with IFN-{gamma} for 24 h; I+P, cells treated with IFN-{gamma} for 24 h in the presence of an inhibitor of JAK, P6. The sizes of PCR products for iNOS and GAPDH were 499 and 226 bp, respectively. B: quantitative analysis of changes in iNOS mRNA expression in response to the 24-h incubation with IFN-{gamma} in the absence (I; n = 5) or presence of P6 (I+P6; n = 5). Values are normalized to that of control cells (C; n = 5). C: representative photographs of fluorescent NO imaging are shown for control (C) and IFN-{gamma}-treated (24 h; I and I+L) cells. In I+L cells, an inhibitor of nitric oxide synthase (NOS), N{omega}-nitro-L-arginine methyl ester (L-NAME), was present during diaminofluoroscein-2 diacetate (DAF-2) loading. Magnification: x200. D: quantitative analysis of the fluorescent intensity for cells shown in C. Twenty cells were analyzed in each group. The doses of IFN-{gamma}, P6, and L-NAME were 20 ng/ml, 1 µM, and 100 µM, respectively. **Significantly different (P < 0.01) compared with control cells.

 
As reported previously, NO had a dose dependently biphasic effect on the inwardly rectifying K+ channel in RPTECs. Namely, a NO donor, SNP, stimulated channel activity at 10 µM and suppressed it at 1 mM (22). Thus we examined how the enhanced production of NO affected channel activity in IFN-{gamma}-treated (24-h) cells, using the cell-attached configuration of the patch-clamp technique. Consistent with our previous reports in control cells (19, 22), a NOS inhibitor, L-NAME, added to the bath at 100 µM suppressed channel activity, whereas a NOS substrate, L-Arg, added at 500 µM stimulated it (Fig. 2A). As shown in Fig. 2D, L-NAME significantly reduced channel activity to 30.5 ± 11.6% of the control, whereas L-Arg caused a significant increase (1.80 ± 0.16-fold) in channel activity. These observations suggested that the endogenous NO in control cells contributed to the maintenance of the basal channel activity and was not so high as to suppress the channel even in the presence of L-Arg. However, in the cells treated with IFN-{gamma} for 24 h, L-NAME activated the channel (Fig. 2B), while L-Arg suppressed it (Fig. 2C). The data on the effects of L-NAME and L-Arg in IFN-{gamma}-treated cells are summarized in Fig. 2D. L-NAME caused a significant increase (1.65 ± 0.23-fold) in channel activity in these cells, although the prolonged exposure of cells to L-NAME reduced channel activity in several patches. L-Arg reduced channel activity to 47.8 ± 19.4% of control. Such responses to NO reagents were in sharp contrast to those in the control cells. These results suggest that NO production was excessive and high enough to suppress channel activity in IFN-{gamma}-treated cells. Thus it is likely that inhibition of NOS suppressed excessive NO production, resulting in the elevation of channel activity. Taken together, IFN-{gamma} would possess a delayed suppressive effect on channel activity, which was dependent on the enhanced iNOS mRNA expression and the consequent increase in NO production.


Figure 2
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  		Fig. 2. Effects of a NOS inhibitor, L-NAME, and a NOS substrate, L-arginine (L-Arg), on channel activity in control or IFN-{gamma}-treated (24 h) cells. A: representative current trace showing that L-NAME suppressed whereas L-Arg stimulated channel activity in control cells. B and C: representative current traces showing effects of L-NAME (B) and L-Arg (C) in cells subjected to 24-h incubation with IFN-{gamma}. In contrast to the control cells, L-NAME stimulated and L-Arg suppressed channel activity. The doses of L-NAME, L-Arg, and IFN-{gamma} were 100 µM, 500 µM, and 20 ng/ml, respectively. These current traces were obtained from separate cell-attached patches at a holding potential (Vp) of 0 mV. Dotted line, closed-channel level. Short thick horizontal bar on the left of each trace, open channel level. D: summary of the effects of L-NAME (n = 10) and L-Arg (n = 10) in control cells (open bars) or L-NAME (n = 12) and L-Arg (n = 12) in IFN-{gamma}-treated cells (filled bars). NPo,e and NPo,c, channel activities under experimental and control conditions, respectively; N, maximum no. of channels observed in the patch; Po, open probability. *P < 0.05 and **P < 0.01, significantly different compared with respective initial control levels.

 
Regarding the mechanisms of the suppressive effect of NO on K+ channel activity, Lu et al. (17) reported that an interaction between NO and superoxide would be important. Although superoxide is converted to hydrogen peroxide (H2O2) by SOD, excessive NO competes with this enzyme for superoxide to form peroxynitrite (OONO–), which might, in turn, cause oxidation and/or nitrosylation of the channel and/or its related proteins. We also examined whether such a mechanism would be involved in the suppressive effect of high NO in RPTECs. Figure 3A shows that 1 mM SNP suppressed channel activity in control cells, which was consistent with our previous report (22). As shown in Fig. 3B, however, the suppressive effect of 1 mM SNP was not observed in the presence of a SOD mimetic, tempol (1 mM). Furthermore, tempol increased channel activity in IFN-{gamma}-treated (24 h) cells (Fig. 3C). These data are summarized in Fig. 3D. In control cells, 1 mM SNP reduced channel activity to 27.5 ± 5.4% of the control. Although tempol itself had no significant effect on channel activity in control cells, it blocked the suppressive effect of 1 mM NO, with the channel activity staying around the control level. In IFN-{gamma}-treated cells, tempol significantly increased channel activity (1.50 ± 0.19-fold). Considering these observations, it was concluded that superoxide would contribute to the suppressive effect of high NO in RPTECs.


Figure 3
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  		Fig. 3. Effect of tempol on the high NO-induced channel suppression. A: representative current trace showing that a NO donor, sodium nitroprusside (SNP), suppressed channel activity in control cells. B: suppressive effect of SNP on channel activity was blocked by a superoxide dismutase mimetic, tempol, in control cells. C: representative current trace showing that tempol activated channel activity in IFN-treated (24-h) cells. SNP and tempol were used at 1 mM. These current traces were obtained from separate cell-attached patches at a Vp of 0 mV. D: summary of the effects of 1 mM SNP (n = 10), tempol (n = 10), and tempol+SNP (n = 10) in control cells (open bars) or tempol (n = 12) in IFN-{gamma}-treated cells (filled bars). *P < 0.05 and **P < 0.01, significantly different compared with respective initial control levels.

 
Next, we examined whether IFN-{gamma} would acutely modulate channel activity in control cells. As shown in Fig. 4A, IFN-{gamma} added to the bath at 20 ng/ml stimulated channel activity in a few minutes in a reversible manner. Since a relatively low concentration of SNP (10 µM) stimulated channel activity (22), it is possible that IFN-{gamma} would acutely cause a moderate increase in NO production, which stimulates channel activity. However, this possibility was negated by the observation that the acute stimulatory effect of 20 ng/ml IFN-{gamma} was not abolished by 100 µM L-NAME (Fig. 4B). These data are summarized in Fig. 4C. IFN-{gamma} caused a significant increase (1.93 ± 0.19-fold) in channel activity. L-NAME significantly reduced channel activity to 27.0 ± 12.0% of control, whereas subsequent addition of IFN-{gamma} caused a 1.37 ± 0.21-fold increase in channel activity compared with control. Thus it seemed likely that the acute stimulatory effect of IFN-{gamma} on channel activity was NO independent.


Figure 4
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  		Fig. 4. Acute effect of IFN-{gamma} on channel activity in control cells and ineffectiveness of L-NAME in blocking the acute effect of IFN-{gamma}. A: representative current trace showing that IFN-{gamma} acutely stimulated channel activity. B: acute stimulatory effect of IFN-{gamma} was not blocked by L-NAME, indicating that the stimulatory mechanism was NO independent. Each trace was obtained from separate cell-attached patches. The doses of IFN-{gamma} and L-NAME were 20 ng/ml and 100 µM, respectively. C: summary of the effects of IFN-{gamma} alone (n = 15), L-NAME alone (n = 7), and L-NAME+IFN-{gamma} (n = 7). **Significantly different (P < 0.01) compared with control cells.

 
Furthermore, we examined acute effects of IFN-{gamma} on NO production in control cells, using fluorescent NO imaging. As shown in Fig. 5A, a 2-min basal recording period was followed by a 10-min experimental recoding period, during which cells were exposed to a vehicle or 20 ng/ml IFN-{gamma}, with the fluorescent signals being acquired every 10 s. The pattern of time-dependent increase in F/F0 induced by IFN-{gamma} was similar to that induced by the vehicle (Fig. 5A). After the experimental recording period, the cells were washed and further exposed to 500 µM L-Arg for 10 min. In both experimental groups, L-Arg caused a greater increase in F/F0, indicating that DAF-2 had successfully been loaded and that the responsiveness of cells was intact (Fig. 5A). Figure 5B summarizes the net increases in F/F0 at 1, 3, and 10 min after the addition of the vehicle or IFN-{gamma}. There was no significant difference in {Delta}F/F0 between the vehicle and IFN-{gamma} at all time points. These observations, as well as the results from patch-clamp experiments, strongly suggested that the acute stimulatory effect of IFN-{gamma} was independent of the moderate activation of the NO pathway.


Figure 5
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  		Fig. 5. Acute effect of IFN-{gamma} on intracellular NO production in control cells. A: representative fluorescent recordings for NO in response to a vehicle (top) or IFN-{gamma} (bottom). In both cases, L-Arg was also tested to confirm the successful DAF-2 loading and functional integrity of cells. B: summary of the net increases in fluorescent intensity at 1, 3, and 10 min after addition of the vehicle (n = 7) or IFN-{gamma} (n = 7).

 
As mentioned above, receptor binding of IFN-{gamma} is known to activate JAK to initiate intracellular signaling cascades. Indeed, IFN-{gamma} enhanced iNOS mRNA expression through activation of JAK in RPTECs. Thus we explored the involvement of receptor binding and activation of JAK in the acute stimulatory effect of IFN-{gamma} in control cells. Figure 6A shows that the IFN-{gamma}-induced channel activation was abolished by the subsequent addition of a nonselective JAK inhibitor, P6, at 1 µM. A JAK2-selective inhibitor, AG490 exhibited a similar effect at 10 µM (Fig. 6B). As shown in Fig. 6C, P6 alone did not affect channel activity, although it blocked the stimulatory action of subsequently added IFN-{gamma}. The data are summarized in Fig. 6D. IFN-{gamma} caused a significant increase (1.91 ± 0.17-fold) in channel activity, which was abolished by P6 and AG490, with the channel activity returning to the control level. P6 alone had no significant effect on channel activity. These results suggested that IFN-{gamma} would acutely stimulate channel activity via its binding to IFNGR and subsequent activation of JAK.


Figure 6
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  		Fig. 6. Effects of JAK inhibitors on the IFN-{gamma}-induced channel activation. A: representative current trace showing that P6 abolished the acute stimulatory effect of IFN-{gamma}. B: another JAK inhibitor, AG490, similarly abolished the acute effect of IFN-{gamma}. C: representative current trace showing that P6 had no apparent effect on the control channel activity by itself, although it suppressed the acute effect of IFN-{gamma}. D: summary of the effects of IFN-{gamma} alone (n = 14), IFN-{gamma}+P6 (n = 7), IFN-{gamma}+AG490 (n = 7), and P6 alone (n = 14). **Significantly different (P < 0.01) compared with respective initial control levels.

 
The activity of the 40-pS K+ channel in RPTECs was upregulated by PKA- and PKG-mediated phosphorylation processes (20, 21). The latter was involved in the stimulatory effect of a low concentration of NO on channel activity (22). Therefore, we examined the possible involvement of PKA and PKG in the acute stimulatory effect of IFN-{gamma}. As shown in Fig. 7, A and B, IFN-{gamma} reactivated the channel suppressed by a PKA-inhibitor, KT5720 (500 nM), or a PKG-inhibitor, KT5823 (1 µM), even in the presence of these inhibitors. These data are summarized in Fig. 7C. KT5720 and KT5823 significantly reduced channel activity to 25.2 ± 8.0 and 22.8 ± 10.0% of initial control levels, respectively. Subsequent addition of IFN-{gamma} restored channel activity, suggesting that the acute stimulatory effect of this cytokine was independent of the PKA- and PKG-mediated phosphorylation.


Figure 7
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  		Fig. 7. Representative current traces showing that IFN-{gamma} reactivated the channel which was suppressed by a PKA inhibitor, KT5720 (A), or a PKG inhibitor, KT5823 (B). These traces were recorded with separate cell-attached patches. The doses of KT5720, KT5823, and IFN-{gamma} were 500 nM, 1 µM, and 20 ng/ml, respectively. C: summary of the effects of KT5720 alone (n = 8), KT5720+IFN-{gamma} (n = 8), KT5823 alone (n = 7), and KT5823+IFN-{gamma} (n = 7). **Significantly different (P < 0.01) compared with the initial control level.

 
Finally, we examined whether PI3K, which was reported to be activated by IFN-{gamma}, was involved in the acute stimulatory effect of this cytokine, using PI3K inhibitors. As shown in Fig. 8, A and B, wortmannin (100 nM) and LY294002 (1 µM) suppressed channel activity, and IFN-{gamma} reactivated the channel even in the presence of these PI3K inhibitors. The data are summarized in Fig. 8C. Wortmannin and LY294002 significantly reduced channel activity to 19.2 ± 6.0 and 25.4 ± 8.0% of initial control levels, respectively. The reduced channel activity was restored by IFN-{gamma} to the initial control levels, suggesting that the acute stimulatory effect of this cytokine was independent of PI3K activity.


Figure 8
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  		Fig. 8. Representative current traces showing that IFN-{gamma} reactivated the channel which was suppressed by PI3K inhibitors, wortmannin (A) and LY294002 (B). These traces were recorded with separate cell-attached patches. The doses of wortmannin, LY294002, and IFN-{gamma} were 100 nM, 10 µM, and 20 ng/ml, respectively. C: summary of the effects of wortmannin alone (n = 6), wortmannin+IFN-{gamma} (n = 6), LY294002 alone (n = 8), and LY294002+IFN-{gamma} (n = 8). **Significantly different (P < 0.01) compared with the initial control level.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The cytokine family consists of many distinct members, each of which modulates a wide variety of cellular functions. It was reported that some proinflammatory cytokines, such as IFN-{gamma}, IL-1, and TNF-{alpha}, altered Na+ transport in the nephron (5, 27). Because transepithelial Na+ reabsorption depends on K+ channel activity, as well as Na+-K+-ATPase, it would be important to know whether cytokines affect the channel. It is also of note that these cytokines are known to promote expression of iNOS (1), which is involved in the regulation of K+ channel activity in cultured human proximal tubule cells (19). However, little information is available regarding the effects of cytokines on K+ channel activity in renal tubular epithelia, excepting a report by Wei et al. (31). They demonstrated that TNF acutely stimulated activity of an apical 70-pS K+ channel in the thick ascending limb of rat kidney (31). In the present study, we revealed that IFN-{gamma} possessed a delayed suppressive effect and an acute stimulatory effect on activity of the 40-pS K+ channel in cultured human proximal tubule cells.

Our data strongly suggest that the delayed suppressive effect of IFN-{gamma} on channel activity was mediated, at least in part, by a high concentration of NO. This notion was based on the following observations. First, NO had a biphasic effect on K+ channels in renal tubular epithelia, as reported previously (17, 22, 30). NO donors stimulated K+ channel activity at low concentrations (<10 µM) and suppressed it at high concentrations (0.1–1 mM) (17, 22, 30). Second, 24-h incubation of cells with IFN-{gamma} resulted increased iNOS mRNA expression and NO production. Third, a NOS inhibitor stimulated channel activity in the cells treated with IFN-{gamma} for 24 h, indicating that NO production was so high as to suppress channel activity in these cells. It was reported that excessive NO reacted with superoxide to form peroxynitrite, which would impair K+ channel activity in rat cortical collecting duct (17). In our study, tempol, which metabolizes superoxide, abolished the channel suppression by 1 mM SNP in control cells and stimulated channel activity in IFN-{gamma}-treated cells, where NO production increased. Therefore, interaction of NO with superoxide and subsequent formation of peroxynitrite would also be important in the delayed suppressive effect of IFN-{gamma} on channel activity in cultured human proximal tubule cells.

It is generally accepted that the biological actions of IFN-{gamma} are mediated by a specific receptor, IFNGR, and the receptor-associated tyrosine kinase, JAK (4). Upon binding of IFN-{gamma} to IFNGR, JAK phosphorylates the signal transducer and activator of transcription (STAT) proteins, which subsequently modulates transcription of various genes. With regard to the promotion of iNOS mRNA expression, it has been demonstrated that IFN-{gamma} utilizes STAT1 and IFN-regulatory factor-1, which binds to the IFN-stimulated response element in the iNOS gene (28, 29). In contrast, the stimulatory effect of IFN-{gamma} on channel activity seemed to be a nongenomic action, since it was observed in a few minutes. However, similar to the delayed genomic action, the acute stimulatory effect of IFN-{gamma} was also mediated by IFNGR and JAK. This notion was supported by the observation that a JAK inhibitor abolished the IFN-{gamma}-induced channel activation. A nonselective JAK inhibitor itself had no apparent effect on channel activity. Therefore, the JAK-mediated signaling would be inactive under the control condition in cultured human proximal tubule cells.

Although it was highly likely that the acute stimulatory effect of IFN-{gamma} was receptor mediated, the downstream mechanisms of JAK signaling are currently unknown. It has been reported that a low concentration of NO stimulates activity of the renal tubular K+ channels through activation of the cGMP/PKG pathway (22, 30). However, inhibitors of NOS and PKG did not block the acute stimulatory effect of IFN-{gamma}. In fact, IFN-{gamma} did not acutely affect intracellular NO production compared with the vehicle. Furthermore, the PKA-mediated phosphorylation, which was also reported to stimulate channel activity (21), was not involved in the acute effect of IFN-{gamma}. Another candidate for the mediator of the acute effect of IFN-{gamma} is PI3K (3, 12, 23). PI3K phosphorylates a membrane phospholipid, phosphatidylinositol 4,5 bisphosphate (PIP2), to PIP3. It is believed that PIP2 plays an important role in stabilizing the open state of inwardly rectifying K+ channels (15, 16). Thus the PI3K-induced depletion of PIP2 might tend to reduce channel activity. Li et al. (14) reported that inhibition of PI3K stimulated ROMK-like K+ channel activity in rat cortical collecting duct. In contrast, PI3K inhibitors reduced channel activity in cultured human proximal tubule cells. The discrepancy of effect of PI3K inhibition on K+ channel activity might result from the different action of PI3K. Indeed, PI3K acts not only as a lipid kinase but also as a serine/threonine protein kinase (3, 12, 23). It was also suggested in our study that PI3K might be involved in the acute stimulatory effect of IFN-{gamma}. However, IFN-{gamma} stimulated channel activity in the presence of PI3K inhibitors, indicating that the acute effect of IFN-{gamma} was PI3K independent. Further studies are required to clarify the precise mechanisms of the acute effect of IFN-{gamma}.

The physiological significance of the effects of IFN-{gamma} on K+ channel activity is also obscure. Rather, the significance should be considered under pathological conditions. Unlike type I IFN secreted by various cell types, IFN-{gamma} is primarily secreted by Th1 lymphocytes and natural killer cells (23, 28). IFN-{gamma} increases in response to microbial infection, causing immunomodulation, apoptosis, or cell injury (23, 28). Some investigators suggested relationships between K+ channel activity and cell injury during ischemia or endotoxemia (24, 25, 32). Thus the IFN-{gamma}-induced cell injury might be related to its effects on K+ channel activity. It is also of note that IFN-{gamma} is sometimes used for the treatment of renal cell carcinoma (2, 18). Considering that it might cause renal cell injury, special attention should be paid to its therapeutic use.

In summary, IFN-{gamma} possessed a delayed suppressive effect, which was dependent on enhanced iNOS expression, and an acute stimulatory effect, which was independent of the NO/cGMP/PKG, cAMP/PKA, and PI3K pathways, on the activity of the inwardly rectifying K+ channel in cultured human proximal tubule cells.


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This work was supported in part by a grant from the Corporation for Private School of Japan (to M. Kubokawa).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Kubokawa, Dept. of Physiology, Iwate Medical Univ. School of Medicine, 19-1 Uchimaru, Morioka, 020-8505 Japan (e-mail: mkubokaw{at}iwate-med.ac.jp)

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.


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