Role of TASK2 in the Control of Apoptotic Volume Decrease in Proximal Kidney Cells

Abstract

Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor α. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-α-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production. Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor α. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-α-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production. Like many epithelial cells, renal cells are capable of regulating their volume in response to variations in external osmotic pressure (1Coca-Prados M. Anguita J. Chalfant M.L. Civan M.M. Am. J. Physiol. 1995; 268: C572-C579Crossref PubMed Google Scholar, 2De Smet P. Simaels J. Van Driessche W. Pflugers Arch. 1995; 430: 936-944Crossref PubMed Scopus (33) Google Scholar, 3Lopes A.G. Amzel L.M. Markakis D. Guggino W.B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2873-2877Crossref PubMed Scopus (26) Google Scholar). Briefly, cells respond to an increase in medium osmolarity by a process referred to as regulatory volume increase, whereas cells respond to the dilution of external medium by a regulatory volume decrease (RVD) 2The abbreviations used are:RVDregulatory volume decreaseAVDapoptotic volume decreaseChTxcharybdotoxinIbTXiberiotoxinSTSstaurosporineCsAcyclosporin ATNF-αtumor necrosis factor-αROSreactive oxygen speciescarboxy-H2DCFDA(5-and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetateTEAtetraethylammoniumNACN-acetylcysteine. (4Pedersen S.F. Mills J.W. Hoffmann E.K. Exp. Cell Res. 1999; 252: 63-74Crossref PubMed Scopus (98) Google Scholar). A variety of transport pathways have been implicated in both processes and result in rapid water flux across the plasma membrane, which causes cells to recuperate their initial volume correspondingly (5Okada Y. Maeno E. Shimizu T. Dezaki K. Wang J. Morishima S. J. Physiol. 2001; 532: 3-16Crossref PubMed Scopus (466) Google Scholar). Along the proximal tubule, the cells are submitted to hypotonic shock because water accompanies the transport of ions by membrane co-transports. In response to this osmotic stress, the proximal cells undergo a RVD process that is characterized by an exit of Cl- and K+ ions, which finally drives water efflux (6Knoblauch C. Montrose M.H. Murer H. Am. J. Physiol. 1989; 256: C252-C259Crossref PubMed Google Scholar). However, changes in cell volume are not only due to variation in medium osmolarity. It is now well established that the initial process leading toward apoptotic cell death is coupled to normotonic cell shrinkage (7Maeno E. Ishizaki Y. Kanaseki T. Hazama A. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9487-9492Crossref PubMed Scopus (660) Google Scholar), called apoptotic volume decrease (AVD). The proximal tubule is a major site of agent-induced nephrotoxicity (drugs, heavy metals, hypoxia etc.), which can induce AVD and lead to cell death by apoptosis. It is therefore interesting to understand the mechanisms involved in this phenomenon. As in RVD, the changes in cell volume during AVD are the consequence of an exit of Cl- and K+ from the cells, and the question arises as to whether the Cl- and the K+ currents are driven by the same type of channels (8Okada Y. Maeno E. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 130: 377-383Crossref PubMed Scopus (169) Google Scholar). The AVD-induced Cl- channel has been identified as a volume-sensitive outwardly rectifying Cl- channel in both HeLa cells and cardiomyocytes treated with staurosporine (9Shimizu T. Numata T. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6770-6773Crossref PubMed Scopus (210) Google Scholar, 10Okada Y. Shimizu T. Maeno E. Tanabe S. Wang X. Takahashi N. J. Membr. Biol. 2006; 209: 21-29Crossref PubMed Scopus (215) Google Scholar). This channel shares many properties with the Cl- channel that are induced in RVD in mouse proximal cells (5Okada Y. Maeno E. Shimizu T. Dezaki K. Wang J. Morishima S. J. Physiol. 2001; 532: 3-16Crossref PubMed Scopus (466) Google Scholar). The molecular nature of this Cl- channel is not fully elucidated, but the literature data converges toward the conclusion that this Cl- channel type is probably ubiquitously expressed in animal cells (7Maeno E. Ishizaki Y. Kanaseki T. Hazama A. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9487-9492Crossref PubMed Scopus (660) Google Scholar). By contrast, the molecular identity of the K+ channel involved in both AVD and RVD is still under discussion because different candidates have been proposed depending on the tissue under study (8Okada Y. Maeno E. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 130: 377-383Crossref PubMed Scopus (169) Google Scholar, 11Burg E.D. Remillard C.V. Yuan J.X. J. Membr. Biol. 2006; 209: 3-20Crossref PubMed Scopus (130) Google Scholar, 12Remillard C.V. Yuan J.X. Am. J. Physiol. 2004; 286: L49-L67Crossref PubMed Scopus (224) Google Scholar, 13Trimarchi J.R. Liu L. Smith P.J. Keefe D.L. Am. J. Physiol. 2002; 282: C588-C594Crossref PubMed Scopus (77) Google Scholar, 14Lu L. Prog. Retin. Eye Res. 2006; 25: 515-538Crossref PubMed Scopus (37) Google Scholar, 15Bock J. Szabo I. Jekle A. Gulbins E. Biochem. Biophys. Res. Commun. 2002; 295: 526-531Crossref PubMed Scopus (63) Google Scholar, 16Montague J.W. Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. Steroids. 1999; 64: 563-569Crossref PubMed Scopus (65) Google Scholar, 17Beauvais F. Michel L. Dubertret L. J. Leukocyte Biol. 1995; 57: 851-855Crossref PubMed Scopus (136) Google Scholar). In a previous study, we demonstrated that TASK2 channels were expressed in kidney proximal cells. These channels were involved in the K+ and Cl- efflux that occurred during RVD. TASK2 belongs to the family of two pore domains K+ channels. Interestingly, Trimarchi et al. (13Trimarchi J.R. Liu L. Smith P.J. Keefe D.L. Am. J. Physiol. 2002; 282: C588-C594Crossref PubMed Scopus (77) Google Scholar) have provided evidence that two-pore domain K+ channels underlie K+ efflux during AVD in mouse embryos. Based on these observations, it was reasonable to postulate that TASK2 K+ channels could also be involved in the regulation of AVD and apoptosis in the proximal tubules. Thus, the present study addresses the role of TASK2 in apoptosis induced by staurosporine. In a large variety of cells, staurosporine is known to induce apoptosis through a mitochondria-mediated pathway and to increase oxidative stress by the production of ROS generated by the mitochondria (9Shimizu T. Numata T. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6770-6773Crossref PubMed Scopus (210) Google Scholar, 18Gil J. Almeida S. Oliveira C.R. Rego A.C. Free Radic. Biol. Med. 2003; 35: 1500-1514Crossref PubMed Scopus (93) Google Scholar). In proximal cells, this mitochondrial mechanism may be the predominant mode of inducing apoptosis in the presence of many nephrotoxic agents (19Thevenod F. Nephron Physiol. 2003; 93: 87-93Crossref Scopus (225) Google Scholar, 20Baek S.M. Kwon C.H. Kim J.H. Woo J.S. Jung J.S. Kim Y.K. J. Lab. Clin. Med. 2003; 142: 178-186Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 21Erkan E. Devarajan P. Schwartz G.J. J. Am. Soc. Nephrol. 2007; 18: 1199-1208Crossref PubMed Scopus (80) Google Scholar). Therefore, the staurosporine-induced apoptosis is a useful model to assess the role of ion channels in controlling apoptosis in renal cells. The present study was conducted on proximal tubule cell lines originating from wild type and task2-/- mice. In wild type mice, we demonstrated that staurosporine-induced AVD was mainly associated with the activity of TASK2 K+ channels. Surprisingly, staurosporine-induced AVD persisted in the task2-/- proximal cell line and could be controlled by a Ca2+-activated K+ channel that is sensitive to iberiotoxin. regulatory volume decrease apoptotic volume decrease charybdotoxin iberiotoxin staurosporine cyclosporin A tumor necrosis factor-α reactive oxygen species (5-and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate tetraethylammonium N-acetylcysteine Transformation of Primary Cultures with pSV3 neo and Culture Protocol—The primary cell culture technique has been described in detail in previous studies (22Belfodil R. Barriere H. Rubera I. Tauc M. Poujeol C. Bidet M. Poujeol P. Am. J. Physiol. 2003; 284: F812-F828Google Scholar). Briefly, 10-day-old primary cultures of S1 and S2 segments of proximal tubules from wild type and task2-/- mice were transfected with pSV3 neo using Lipofectin (Invitrogen). After 48 h, selection of the clones was performed by the addition of G418 (500 μg/ml). Culture medium (Dulbecco's modified Eagle's medium-F12, Sigma, Saint Quentin Fallavier, France) containing 250 μg/ml G418, 15 mm NaHCO3, 20 mm HEPES (pH 7.4), growth factors (23Barriere H. Rubera I. Belfodil R. Tauc M. Tonnerieux N. Poujeol C. Barhanin J. Poujeol P. J. Membr. Biol. 2003; 193: 153-170Crossref PubMed Scopus (35) Google Scholar), and 1% FCS was changed every day. Resistant clones were isolated, subcultured, and used after 10 trypsinization steps. Immortalized proximal wild type and task2-/- cell lines were grown on collagen-coated supports (35-mm Petri dishes) in a 5% CO2 atmosphere at 37 °C in the culture medium described above. Apoptosis Induction—Apoptosis was induced by staurosporine (STS, 1 μm), cyclosporin A (CsA, 25 μm), or tumor necrosis factor-α (TNF-α, 0.5 ng/ml) in proximal wild type and task2-/- cell lines that were maintained in serum and growth factor-free culture medium (Dulbecco's modified Eagle's medium/F-12, Sigma) in a 5% CO2 atmosphere at 37 °C. Staurosporine (STS) and cyclosporin A (CsA) were dissolved in Me2SO. The quantity of Me2SO added to the incubation solutions never exceeded 0.1%. Control experiments were performed by incubating the cells with 0.1% Me2SO only. Measurement of Caspase-3 Activity—Caspase-3 activity was measured using colorimetric assays (CaspACE™ assay system, colorimetric, Promega). The activity was assayed in triplicate or quadruplicate on protein extracts obtained after lysis of transformed proximal wild type and task2-/- cells. As indicated by the supplier, the involvement of other related proteases was excluded by observing the difference between color intensity in the absence and presence of a specific caspase-3 inhibitor (Z-VAD). The absorbance was measured at 405 nm using an Automated Microplate Reader ELX-800 (Bio-Tek Instruments, Inc.). Apoptotic Cell Counts—STS-induced apoptosis was studied in wild type and task2-/- cell lines. Cells were grown in 35-mm Petri dishes. After an appropriate incubation with the apoptosis inductor (STS), living cells were carefully washed with fresh culture medium and incubated 10 min in the presence of Hoechst-33258 (50 μm) and propidium iodide (1 μm). Digital micrographs were successively taken at 455 nm for Hoechst-33258 and 585 nm for propidium iodide. Afterward, the cell preparation was washed and stained with orcein solution (250 mg of orcein, 2 ml of ethanol 70%, 150 μl of HCl, 12 n). Micrographs of orcein-stained cells were then taken. Thus in a given culture, the same zone was visualized after individual staining with Hoechst-33258, propidium iodide, and orcein. Apoptotic cells were counted by comparing the three stains. A cell was considered apoptotic only if the nucleus was not stained by propidium iodide and presented chromatin condensation with visible apoptotic bodies. The counts of apoptotic nuclei were performed directly on the digital micrographs. Between 100 and 200 cells were scored by three different observers who were blinded to the culture conditions. The numbers of cells with DNA condensation and propidium iodide staining were expressed as the percentage of total cells. Cell Volume Measurement—Cell volume was measured by an electronic sizing technique using a CASY 1 cell counter (SCHÁRFE SYSTEM®). Briefly, proximal wild type and task2-/- cells that were exposed to different treatments (STS, CsA, or TNF-α) were rapidly trypsinized (1 times for 45 s), and cell volume measurement was performed just after suspending the cells in Casyton® solution (NaCl isotonic solution). Electrophysiological Studies—Whole cell currents were performed on cultured proximal wild type and task2-/- cells grown on 35-mm Petri dishes maintained at 37 °C for the duration of the experiments. The ruptured whole cell configuration of the patch-clamp technique was used. Patch pipettes (2- to 4-megaohm resistance) were made from borosilicate capillary tubes (1.5 mm outer diameter, 1.1 mm inner diameter; Fisher Manufacturing) using a two-stage vertical puller (model PP 830, Narishige, Tokyo, Japan). Cells were observed using an inverted microscope; the stage of the microscope was equipped with a water robot micromanipulator (model WR 89, Narishige). The patch pipette was connected via an Ag-AgCl wire to the head stage of a patch amplifier (model VP 500, Biologic). The membrane was ruptured by additional suction to achieve the conventional whole cell configuration. Settings available on the amplifier were used to compensate for cell capacitance. The series resistances were not compensated, but experiments in which the series resistance was higher than 20 megaohms were discarded. The offset potentials between both electrodes were zeroed before sealing, and the liquid junction potential was measured experimentally prior to each experiment and corrected accordingly (measured junctions potentials were 11.34 ± 0.79 mV for K+ conductance experiments). Solutions were perfused in the extracellular bath using a four-channel glass pipette, with the tip placed as close as possible to the clamped cell. Voltage-clamp commands, data acquisition, and data analysis were controlled via the VP 500 amplifier connected to a computer. The whole cell currents resulting from voltage stimuli were sampled at 2.5 kHz and filtered at 1 kHz. Cells were held at -50 mV, and 400-ms pulses from -100 to +120 mV were applied in 20-mV increments. The pipette solution contained (in mm): 100 K-gluconate, 25 KHCO3, 20 KCl, 10 HEPES (pH 7.4 adjusted with 1 n KOH), 5 MgATP, and 0 or 30 EGTA (Pos = 300 milliosmole/kg of H2O). To avoid spontaneous activation of volume-sensitive K+ currents, the bath solution was slightly hyperosmotic and contained (in mm): 110 NMDG-Cl, 5 glucose, 5 potassium gluconate, 1 CaCl2, 1 HEPES (pH 7.4 adjusted with 1 n HCl), and 100 mannitol (Pos = 330-340 milliosmole/kg of H2O). Measurement of Reactive Oxygen Species (ROS)—Levels of cellular oxidative stress were measured using the fluorescent probe (5-and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). Carboxy-H2DCFDA is a cell-permeable indicator for ROS that becomes spontaneously fluorescent when the acetate groups are removed by intracellular esterases and cell oxidation. This probe is trapped mainly in the cytoplasm and is oxidized by several ROS, most notably hydrogen peroxide. Briefly, proximal wild type and task2-/- cells were incubated in Petri dishes at 37 °C for 30 min in the presence of carboxy-H2DCFDA (10 μm) and gently washed in serum-free culture medium. Two experimental protocols were developed to measure the fluorescence increase according to the time kinetic leading to ROS increase. For rapid kinetic (less than 1 h), cells were rapidly trypsinized (×10 for 45 s) and incubated in the absence or presence of either STS (1 μm) or NAC (N-acetylcysteine, 10 mm) or both substances. Variations of fluorescence of the cell suspension were measured every 2 min using a Genius Spectrofluorimeter (SAFAS, Monaco) at 538 nm. For long time kinetic (experiments performed in the presence of TNF-α), the ROS production was monitored by using fluorescent video microscopy. Briefly, proximal cell lines grown in 35-mm Petri dishes were incubated in the presence of carboxy-H2DCFDA (10 μm) at 37 °C for 30 min in a humidified atmosphere of 5% CO2, 95% air. Cells were gently washed and incubated in an isotonic serum-free medium containing 30 mm HEPES in the absence or presence of TNF-α. The variation of fluorescence was measured every 15 min (during 24 h) at 538 nm. Data are expressed as the fluorescence ratio F/F0, where F is the relative fluorescence intensity measured every 15 min and F0 the relative fluorescence intensity measured at = The of ROS production were obtained from analysis of cells in each intracellular was measured in cells grown in Petri dishes and for 45 min at in the presence of μm) and The cells were washed with a solution containing in 5 KCl, 1 1 CaCl2, 5 glucose, and 20 HEPES (pH Cells were successively at and nm and the were was the result of an of to The was every 10 For each was monitored in cells. The of et al. M. J. Biol. Full Text PDF PubMed Scopus (80) Google Scholar) was used to from the and were in Me2SO and used at of 1 and 25 was in water and used at a of 0.5 was at 10 mm in a solution containing and were a from Barhanin solutions of (50 mm) and (50 were and at °C and The fluorescent probe carboxy-H2DCFDA was used at 10 solution at 10 mm in was dissolved at mm in Me2SO and added to the solution at a of 2 with NAC (10 tetraethylammonium 1 charybdotoxin 10 and iberiotoxin 100 were obtained from of Caspase-3 and by STS in and task2-/- Cell experiments performed on proximal cell lines have established a role for the TASK2 K+ channel in the regulatory volume decrease during hypotonic a specific cell volume decrease (AVD) is observed during apoptosis In the present study, the involvement of TASK2 channels during induced apoptosis was For this proximal cell lines from wild type and task2-/- mice were exposed to STS (1 μm) for h, and the caspase-3 activity was As in STS induced a increase in caspase-3 activity in wild type cells. Interestingly, this increase was higher than that observed in task2-/- cells This that TASK2 channels were involved in the apoptotic The caspase-3 activation was inhibited by or external the involvement of TASK2 have been to TASK2 K+ In these cells, the of K+ not the caspase-3 Surprisingly, the addition of STS still the of caspase-3 in task2-/- cells. As this increase was not modified by the addition of or by the of the external However, the of or caspase-3 These in task2-/- cells, the caspase-3 activation could be driven by an K+ To these observations, the apoptosis was also on the of type and task2-/- cell lines were stained with and propidium iodide to between apoptotic and cells. the nuclei of wild type cells excluded propidium iodide and exhibited a with the In contrast, after STS (1 h), staining that several cells exhibited staining of and chromatin and were not stained with propidium iodide, of the plasma membrane The condensation and of DNA that STS induced apoptosis. than ± of the total cells exhibited propidium These characteristics could also be observed with orcein cells not chromatin By contrast, a and of of chromatin could be observed after STS and orcein staining not on task2-/- cells a decrease of and chromatin as with wild type cells. the of these the of apoptotic cells was in both cell lines. the percentage of apoptotic cells after or in the presence STS (1 μm). In both cell lines, the percentage of apoptotic cells with the time of each time and h), the percentage of apoptotic cells was higher in wild type than in task2-/- cell lines. h, the percentage of apoptotic cells ± in wild type cell lines but only ± in task2-/- cell lines. the same time h), the percentage of cells ± in the task2-/- cell but only ± in wild type cells AVD in Cell whether the apoptotic process was related to an AVD the time of relative cell volume variation during STS was measured in proximal cell lines from wild type and task2-/- mice. In both cell lines, cell shrinkage as as 1 after STS (1 after of the relative cell volume by ± in wild type cell lines but only by ± in task2-/- cell lines. The AVD was then studied in the presence of or As in in wild type cell lines, the AVD was strongly inhibited by but not by or AVD was by the of the external solution In task2-/- cell lines, the AVD was inhibited by or and was to or external STS Two of K+ in and task2-/- Cell experiments the involvement of the TASK2 channel in the AVD process in wild type proximal cells. Whole cell experiments were then performed to the nature of the K+ conductance by STS the K+ currents in wild type proximal cells before the addition of the voltage outwardly rectifying currents. The indicated a potential of ± 5 mV and a conductance between and +120 of ± = STS induced a large increase of the currents, with a conductance of ± = of the potential ± To determine the possible role of in the of currents, experiments were performed using a pipette solution containing the EGTA mm) to the intracellular In of the cells the conductance was not These K+ currents were inhibited by (10 conductance ± n = the currents at mV under different experimental the currents exhibited to or but were strongly reduced in the presence of or when an external bath solution (pH These characteristics to TASK2 K+ Whole cell experiments were also performed in the task2-/- cell line to the K+ involved in the residual AVD described in the K+ currents in task2-/- proximal cells before and after STS the voltage outwardly rectifying K+ currents conductance = ± = ± 2.5 mV, n = STS the currents, which a conductance of ± ± 2.5 mV, n = of the These K+ were inhibited by the addition of or to the external were also performed using pipette solution containing EGTA mm) to the intracellular In of the cells the conductance was = As in the K+ currents (measured at exhibited a different as with wild type were inhibited by and but to or with an external bath solution (pH These properties of K+ channel were in both cell using the currents in the presence of Thus, the currents at mV were in task2-/- cells ± n = than in wild type cells ± n = this difference by the of the in wild type and task2-/- cell lines. The individual were in 5 groups according to the at mV in the presence of In wild type cells, the K+ currents measured at mV were ± = and only ± in task2-/- cells = The difference was by in the wild type cell of the currents are between and In contrast, in the task2-/- cell line only of the are in this of currents. Moreover 12 are by STS in the task2-/- cell