Effects of chloride channel inhibitors on H2O2-induced renal epithelial cell injury

Xianmin Meng and W. Brian Reeves

Division of Nephrology, University of Arkansas for Medical Sciences and John L. McClellan Memorial Veterans Hospital, Little Rock, Arkansas 72205


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oxidative stress contributes to renal epithelial cell injury in certain settings. Chloride influx has also been proposed as an important component of acute renal epithelial cell injury. The present studies examined the role of Cl- in H2O2-induced injury to LLC-PK1 renal epithelial cells. Exposure of LLC-PK1 cells to 1 mM H2O2 resulted in the following: depletion of intracellular ATP content; DNA damage; lipid peroxidation; and a loss of membrane integrity to both small molecules, e.g., trypan blue, and macromolecules, e.g., lactate dehydrogenase (LDH), and cell death. Substitution of Cl- by isethionate or the inclusion of certain Cl- channel blockers, e.g., diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino)· benzoate (NPPB), and niflumic acid, prevented the H2O2-induced loss of membrane integrity to LDH. In addition, the H2O2-induced loss of membrane integrity was prevented by raising the osmolality of the extracellular solutions, by depletion of cell ATP, and by inhibitors of volume-sensitive Cl- channels. However, these maneuvers did not prevent the H2O2-induced permeability to small molecules or H2O2-induced ATP depletion, DNA damage, lipid peroxidation, or cell death. These results support the view that volume-sensitive Cl- channels play a role in the progressive loss of cell membrane integrity during injury.

oxidant stress; chloride channels; LLC-PK1 cell; necrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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A CARDINAL FEATURE of the necrotic form of cell death is a loss of membrane integrity. Indeed, the development of membrane permeability to small dyes, such as trypan blue or propidium iodide, or to macromolecules, such as lactate dehydrogenase (LDH), has been considered a marker of irreversible necrotic cell death. The mechanisms that are responsible for the loss of membrane integrity are poorly understood. The maintenance of cellular ion homeostasis requires the precise balance of solute influx and efflux via both active and passive membrane transport pathways. It has been proposed that the uptake of solute, mainly sodium and chloride, with subsequent cell swelling contributes to membrane injury in renal epithelial cells (23) and other tissues (6, 12, 18). In support of this view are both in vivo (24) and in vitro studies (27, 35) that demonstrate an increase in cell chloride content and cell volume following ischemia or "chemical hypoxia." However, the role of Cl- influx in subsequent membrane injury and the pathways by which Cl- enters the cell remain uncertain.

Cl- uptake by isolated proximal tubules increases during hypoxia and chemical ATP depletion (27, 31, 43). That Cl- channels are involved in this Cl- uptake is suggested by the observation that certain classic Cl- channel blockers reduce injury in in vitro models of renal tubular injury (31, 41, 43). Miller and Schnellmann (28) have reported the presence of the beta -subunit of the neuronal glycine receptor in the proximal tubule and speculated that the Cl- uptake during cell injury proceeded through these glycine-gated Cl- channels. In a similar vein, Venkatachalam et al. (41) and Waters et al. (42) showed that certain agonists and antagonists of GABA receptors (also a ligand-gated Cl- channel) were cytoprotective in MDCK cells and rabbit proximal tubule cells. However, despite these observations, the role of Cl- in necrotic cell death remains controversial. For example, no in vitro study has shown a protective effect of substituting Cl- with other impermeant anions. Moreover, the fact that both agonists as well as antagonists of the glycine and GABA receptors are cytoprotective suggests that the cytoprotection may be independent of Cl- influx. Finally, Chen and Mandel (11) found that cell swelling and electrolyte fluxes were, in themselves, insufficient to account for anoxia-induced lysis of rabbit proximal tubule cells. Finally, in all of the aforementioned studies, the evaluation of chloride channel inhibitors has been limited to their short-term effects on membrane integrity (either LDH release or vital dye exclusion). No published study has evaluated the effects of chloride channel inhibitors on long-term cell survival.

The present studies examined the role of extracellular Cl- and Cl- channels in oxidant-induced cell injury. LLC-PK1 cells, a model proximal tubule cell line, were exposed to exogenous hydrogen peroxide (H2O2). Exposure to H2O2 produced ATP depletion, lipid peroxidation, DNA damage, and loss of membrane integrity to macromolecules. Replacement of Cl- with impermeant anions or addition of Cl- channel blockers prevented the H2O2-induced loss of membrane integrity without affecting the other manifestations of injury. In addition, Cl- channel blockers did not improve overall cell survival after oxidant injury. Maneuvers that inhibit volume-sensitive Cl- channels also reduced the H2O2-induced loss of membrane integrity. The results support the view that Cl- influx via volume-sensitive Cl- channels contributes to the loss of membrane integrity during oxidant injury but that other cellular targets of oxidant injury determine the ultimate outcome.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Cell culture. LLC-PK1 obtained from the American Type Culture Collection (CRL 1392; ATCC, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mM glutamine (GIBCO, Life Technologies) at 37°C and aerated with 5% CO2-95% air. Cells were grown in either T-75 flasks (Costar) or 12-well plates and studied 1-2 days after reaching confluence.

Oxidant-induced injury. Monolayers were washed free of medium and incubated in a bicarbonate-buffered saline solution (KRB) containing (in mM) 115 NaCl, 25 NaHCO3, 3.5 KCl, 1 KH2PO4, 1.25 CaCl2, and 1 MgSO4 (pH 7.4 after bubbling with 95% O2-5% CO2). For ion-substitution studies, NaCl was replaced by either NaBr, NaNO3, sodium gluconate, or sodium isethionate. Where indicated, hydrogen peroxide was added to a final concentration of 1 mM from a 100 mM stock solution prepared fresh each day.

LDH release. At the indicated times, the incubation medium was removed and the cells were lysed in 0.2% Triton X-100. The activity of LDH in the incubation solution (supernatant) and in the detergent extract of cells was determined spectrophotometrically from the oxidation of NADH (7). The results are expressed as the percentage of the total LDH content of the well which appeared in the supernatant (LDHsuper): percent release = LDHsuper/(LDHsuper + LDHcell) × 100. None of the test agents or vehicles interfered with the assay for LDH.

Trypan blue staining. At the end of the incubation period, the incubation solution was removed and the cells were stained with 0.4% trypan blue in PBS for 5 min followed by two washes with PBS. The cells were then scraped from the dish into 500 µl of KRB. The number of stained and unstained cells was counted using a hemocytometer.

DNA damage. DNA damage was determined using the alkaline unwinding assay (8) as reported previously from this laboratory (33). In this assay, the rate of DNA unwinding under mild alkaline conditions is increased by the presence of either single- or double-strand DNA breaks. The amount of residual double-stranded DNA after alkaline treatment is expressed as a percentage of the total DNA in the sample.

ATP content. At the end of the incubation period, the incubation solution was removed quickly, and the cell monolayer was extracted into 400 µl of cold 2% perchloric acid. The perchloric acid extract was neutralized with KOH and then diluted 500-fold with 10 mM Tris (pH 7.5). The ATP content of the diluted sample was determined by mixing 20 µl of sample with 100 µl of a 10 mg/ml solution of luciferin-luciferase (Sigma Chemical) while measuring the luminescence in a Turner model TD-20e luminometer. The ATP content is expressed relative to the protein content of the well.

Lipid peroxidation. Lipid peroxidation was measured by the thiobarbituric acid (TBA) reaction (13). Cells were incubated for 1 h with or without H2O2 and the other test agents. At the end of the incubation, the supernatant was removed and mixed with an equal volume of 0.76% TBA in 0.25 M HCl and heated at 95°C for 30 min. The samples were cooled to room temperature, and the absorbance at 532 nm was measured in a spectrophotometer. A standard curve was constructed using tetramethoxypropane hydrolyzed by H2SO4 as the substrate for the TBA reaction. The results are expressed as nanomoles of TBA-reactive substances (TBARS) per milligram cell protein.

Chromium-51 release. Confluent cells growing in 12-well tissue culture plates were loaded with 51Cr by overnight incubation with 0.5 µCi 51Cr/well in culture medium. Immediately prior to the experiment, the cells were washed four times with PBS to remove the extracellular 51Cr. The cells were then incubated with the test solutions for 1 h at 37°C. At the end of the incubation, the supernatant solution was removed and the cell monolayer was solubilized in 0.2% Triton X-100. The 51Cr content of the supernatant solution and of the cell extract were determined by scintillation counting. The release of 51Cr is expressed as the percentage of total 51Cr that appeared in the supernatant solution.

MTS assay. Cell survival was quantified using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay (CellTiter 96 Aqueous NonRadioactive Cell Proliferation Assay; Promega, Madison, WI) following the manufacturer's instructions. For this assay, LLC-PK1 cells were grown in 96-well tissue culture plates until confluent. Cells were then treated for 30 min at 37°C in KRB or KRB containing 1 mM H2O2 in the presence or absence of chloride channel inhibitors. After the treatment, the KRB solution was replaced with culture medium, and the plates were returned to the incubator. Cell survival was measured 24 h after the initial treatment using the MTS assay. Briefly, 20 µl of the MTS solution were added to each well, and the plates were incubated for 2 h at 37°C. The absorbance at 490 nm was measured using a microplate reader (Vmax Kinetic Microplate Reader; Molecular Devices, Sunnyvale, CA) and corrected for background absorbance.

Statistical analysis. Values are presented as means ± SE, unless indicated otherwise. Comparisons between data were made using an unpaired t-test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Hydrogen peroxide was used to produce oxidant injury in LLC-PK1 cells. Figure 1 shows the time course of injury, as measured by the release of LDH, after the addition of 1 mM H2O2 to the medium. After 1 h, there was little detectable release of LDH. However, LDH release increased progressively over the next 3 h. The 2-h time point was chosen for many of the subsequent studies.


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Fig. 1.   Time course of H2O2-induced lactate dehydrogenase (LDH) release from LLC-PK1 cells. Confluent LLC-PK1 cells were incubated in either Krebs-Ringer bicarbonate (KRB) or KRB containing 1 mM H2O2. At the indicated times, samples of supernatant solution were removed for measurement of LDH activity. Results are means ± SE for n = 4-20 experiments. *P < 0.0002 vs. control.

To examine the role of extracellular Cl- in oxidant injury, cells were incubated with H2O2 in a solution in which all but 2.5 mM of the Cl- was replaced by isethionate. As shown in Fig. 2, in cells incubated in the standard KRB solution, H2O2 increased the release of LDH from 4 ± 1 to 37 ± 7% (n = 4) whereas in cells incubated in the Cl--free KRB solution, H2O2 increased the LDH release from 5 ± 1 to 14 ± 3% (n = 4). Thus the H2O2-induced release of LDH was significantly diminished in the absence of extracellular Cl- (P < 0.02). Similar results were obtained when Cl- was replaced by gluconate or methanesulfonate (not shown). Figure 3 shows the relation between the extracellular Cl- concentration and H2O2-induced LDH release. There was a clear monotonic increase in LDH release with increasing extracellular Cl- concentrations.


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Fig. 2.   Effects of Cl- substitution on H2O2-induced LDH release. LLC-PK1 cells were incubated in either standard KRB (left) or low-Cl- KRB (right) in presence or absence of 1 mM H2O2. Here, as in Figs. 4-11, open bars represent cells incubated in KRB only, and solid bars represent cells incubated in KRB containing 1 mM H2O2. LDH release was measured after 2 h. Results are means ± SE for 4 experiments. *P < 0.02 vs. high-Cl- H2O2.



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Fig. 3.   Cl- dependence of H2O2-induced LDH release. LLC-PK1 cells were incubated with 1 mM H2O2 for 2 h in solutions in which Cl- concentration was varied by substitution with isethionate. Results are normalized to LDH released into standard KRB solution (containing 120 mM Cl-); n = 3. *P < 0.05 vs. 120 mM Cl-.

We reported previously that certain Cl- channel blockers reduced hypoxia-induced LDH release from freshly isolated proximal tubule suspensions (31). We examined the effects of Cl- channel blockers on the oxidant-induced release of LDH from LLC-PK1 cells. As in hypoxic injury, oxidant-induced LDH release was significantly reduced by a number of Cl- channel blockers (Fig. 4). The overall pattern of protection, i.e., protection by diphenylamine-2-carboxylate (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), but not by stilbenes, was also similar to that seen in hypoxic injury (31, 43).


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Fig. 4.   Effects of Cl- channel blockers on H2O2-induced LDH release from LLC-PK1 cells. LDH release was measured after 2-h incubation in KRB (open bar) or KRB containing 1 mM H2O2 (solid bars) in presence of indicated agents. Concentrations of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), indanyloxyacetic acid (IAA, 94/95), and dinitrostilbene disulfonic acid (DNDS) were 100 µM. Concentration of niflumic acid (NFA) was 400 µM. Concentration of diphenylamine-2-carboxylate (DPC) was 1 mM, and concentration of glycine was 5 mM; n = 31 for control and H2O2, and n = 3-10 for each test agent. *P < 0.03 vs. H2O2 alone.

The ATP content of cells was measured after 1 h of exposure to 1 mM H2O2, a time point prior to the release of significant amounts of LDH (Fig. 1). As noted by other investigators (1, 2), H2O2 resulted in marked intracellular ATP depletion (28.7 ± 5.8 vs. 9.8 ± 2.0 nmol ATP/mg protein, P = 0.02, n = 4). However, as shown in Fig. 5, treatment with either 100 µM NPPB, 1 mM DPC, or 100 µM niflumic acid did not protect cells against the H2O2-induced ATP depletion. In fact, the ATP levels in NPPB-treated cells (3.4 ± 0.3 nmol/mg protein, n = 3, P = 0.04 vs. H2O2) were lower than in cells treated with H2O2 alone.


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Fig. 5.   Effects of H2O2 and Cl- channel blockers on ATP content of LLC-PK1 cells. ATP content was measured after 1-h incubation with 1 mM H2O2 in presence or absence of indicated agents. Concentrations of NPPB, DPC, and NFA were 100 µM; n = 3-4 for each agent. *P < 0.02 vs. control. #P = 0.04 vs. H2O2 alone.

Oxidant stress results in damage to DNA (1, 38). We determined the effects of H2O2 and certain chloride channel blockers on DNA damage using the alkaline unwinding assay (Fig. 6). Cells were exposed to 1 mM H2O2 for 10 min in the presence or absence of either 100 µM NPPB or 1 mM DPC. Even this brief exposure to H2O2 resulted in a significant degree of DNA damage (77 ± 5 vs. 40 ± 3% residual double-strand DNA, n = 6, P < 0.001). However, neither NPPB nor DPC attenuated the H2O2-induced DNA damage (41 ± 9 and 37 ± 5% residual double-strand DNA, respectively, n = 7).


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Fig. 6.   Effects of Cl- channel blockers on H2O2-induced DNA damage. LLC-PK1 cells were incubated with 1 mM H2O2 for 10 min in presence of indicated agents. DNA damage was measured using the alkaline unwinding assay as described in MATERIALS AND METHODS. Residual double-strand DNA (DS DNA) content was significantly reduced by H2O2 (40 ± 3 vs. 77 ± 5%, P < 0.001, n = 7). NPPB and DPC did not prevent H2O2-induced DNA damage (n = 7).

Hydrogen peroxide exposure results in peroxidation of membrane lipids (1, 13). To determine whether chloride channel blockers may have direct antioxidant effects, lipid peroxidation was measured in cells exposed to H2O2 in the presence or absence of the channel blockers. As shown in Fig. 7, treatment of cells for 1 h with 1 mM H2O2 resulted in a large increase in lipid peroxidation products measured as TBARS (0.2 ± 0.2 vs. 1.9 ± 0.1 nmol TBARS/mg protein, P < 0.0001, n = 3). However, the H2O2-induced lipid peroxidation was not significantly altered by any of the agents tested.


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Fig. 7.   Effects of Cl- channel blockers on H2O2-induced lipid peroxidation. LLC-PK1 cells were incubated with 1 mM H2O2 (solid bars) for 1 h in presence of indicated agents. Concentrations of IAA, NPPB, NFA, and DNDS were 100 µM. Concentration of DPC was 1 mM. Peroxidation products were measured using the thiobarbiturate assay and are expressed as nanomoles of thiobarbiturate-reactive substances (TBARS) per milligram protein. Data are means ± SD. *P < 0.01 vs. control; n = 3.

The results shown in Figs. 2-4 indicate that Cl- channel blockers and Cl- substitution decrease LDH release from oxidatively injured cells. LDH is a large cytosolic protein (mol wt = 136,000). As will be discussed later, there is evidence that during injury cells develop size-selective defects in membrane integrity. Therefore, it was relevant to determine whether Cl- channel blockers maintained the membrane integrity for small molecules as they did for LDH. Figure 8 shows that hydrogen peroxide increased the number of cells that could not exclude the vital dye trypan blue (mol wt = 961). Notably, neither 100 µM NPPB nor 5 mM glycine, both of which reduced oxidant-induced LDH release (Fig. 4), reduced oxidant-induced trypan blue uptake. Similarly, hydrogen peroxide increased the release of 51Cr from LLC-PK1 cells (10 ± 1% vs. 40 ± 4%, P < 0.001) but this increased Cr release was not prevented by NPPB or dinitrostilbene disulfonic acid (DNDS) (Fig. 9). Thus Cl- channel blockers prevent the development of membrane permeability to large, but not small, molecules following oxidant injury.


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Fig. 8.   Effects of NPPB and glycine on trypan blue uptake. LLC-PK1 cells were incubated for 2 h in KRB (open bar) or KRB containing 1 mM H2O2 (solid bars) and either NPPB or glycine. *P < 0.0001 vs. control; n = 3-5.



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Fig. 9.   Effects of NPPB and DNDS on H2O2-induced 51Cr release. LLC-PK1 cells were loaded with 51Cr and then incubated for 2 h in KRB (open bar) or KRB containing 1 mM H2O2 (solid bars) and either 100 µM NPPB or 100 µM DNDS. *P < 0.001 vs. control; n = 6.

The effects of Cl- channel blockers on cell survival after oxidant injury were determined by the reduction of MTS, a measure of mitochondrial function. Figure 10 shows the results of experiments in which LLC-PK1 cells were treated with 1 mM H2O2 for 30 min in the presence or absence of Cl- channel blockers or glycine and then incubated in serum-containing culture medium for 24 h. Cell viability at the end of the 24-h incubation was dramatically reduced by the H2O2 exposure. None of the Cl- channel blockers enhanced cell viability. Likewise, 5 mM glycine, which reduced LDH release by ~50% (Fig. 4) had no effect on cell viability.


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Fig. 10.   Effects of Cl- channel blockers on cell viability. Cell viability was determined by the MTS assay as described in METHODS. The value of the absorbance at 490 nm (A490) is proportional to the number of viable cells. LLC-PK1 cells were treated for 30 min in KRB (open bar) or KRB containing 1 mM H2O2 (solid bars) in absence or presence of either 100 µM NPPB, 1 mM DPC, 100 µM NFA, or 5 mM glycine. *P < 0.001 vs. control. Data are means ± SD of n = 6 wells for each agent. Results are representative of 3 experiments.

The data in Figs. 2-4 are consistent with the view that Cl- entry, probably via a Cl- channel, is required to manifest oxidant-induced membrane injury. A variety of Cl- channels are present in the plasma membrane of renal epithelial cells (32), and the channel responsible for Cl- entry during oxidant injury is not known. Cl- channels may be distinguished by their differing anion selectivity, sensitivity to inhibitors, and physiological regulation. Experiments were performed to examine the ability of other anions to support H2O2-induced cell injury. LDH release was measured using solutions in which Cl- was replaced by either NO-3, F-, Br-, or acetate. The H2O2-induced release of LDH (relative to Cl-) for these anions was: NO-3 (2.1) > Br- (1.4) > Cl- (1) > F- (0.8) > acetate (0.25) (n = 3 for each). The basal release of LDH was not affected by any of the anion substitutions. The rank order of these various anions in promoting H2O2-induced LDH release is the same as the permeability of these anions through volume-sensitive Cl- channels (36).

Volume-sensitive anion channels are present in many tissues, including renal epithelial cells (34, 36). These channels are activated by cell swelling and are permeable to Cl- and small organic anions. Figure 11 shows the results of experiments in which the extracellular osmolality was increased by the addition of relatively impermeant solutes. Mannitol, raffinose, and maltose all significantly reduced the H2O2-induced release of LDH. Volume-sensitive anion channels are also blocked by certain nonclassic Cl- channel blockers, such as ketoconazole and tamoxifen (25, 46), and also require intracellular ATP for channel activation (21). Figure 12 shows that 50 µM ketoconazole, 20 µM tamoxifen, and depletion of ATP with 10 µM antimycin A all significantly reduced H2O2-induced release of LDH.


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Fig. 11.   Effects of extracellular hypertonicity on H2O2-induced LDH release. LDH release was measured after 2-h incubation in KRB (open bar) or KRB containing 1 mM H2O2 (solid bars) and 200 mM of either mannitol, raffinose, or maltose. *P < 0.03 vs. H2O2 alone. Data are means ± SE for n = 4-9 experiments.



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Fig. 12.   Effects of inhibitors of volume-sensitive anion channels on H2O2-induced LDH release. LDH release was measured after 2-h incubation in KRB (open bar) or KRB containing 1 mM H2O2 (solid bars) in presence of indicated agents; ketocon., ketoconazole. *P < 0.005 vs. H2O2 alone. Data are means ± SE for n = 3-6 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies examined the role of chloride in oxidative injury to renal epithelial cells. The generation of hydrogen peroxide has been implicated in the pathogenesis of several forms of acute tubular cell injury (3). Whereas several lines of evidence support the view that Cl-, or Cl- transport, may contribute to cell injury induced by hypoxia (31, 43) or ATP depletion (41), the role of Cl- in oxidant-induced injury has not been determined. Moreover, even in hypoxia or ATP depletion, the role of Cl- in cell injury is not firmly established. For example, although certain Cl- channel blockers reduced LDH release from proximal tubules or MDCK cells subjected to hypoxia (31, 43) or ATP depletion (41), substitution of Cl- by other anions has not been shown to confer protection (41). The present studies were intended to provide additional evidence for the role of Cl- in cell injury, to determine whether a role for Cl- can be extended to oxidant-induced injury, and to begin to define the pathways for Cl- entry during injury. In addition, the present studies examined, for the first time, the effects of Cl- channel inhibitors on long-term cell survival following injury.

Exposure of LLC-PK1 cells to hydrogen peroxide in vitro has been shown to result in ATP depletion, lipid peroxidation, DNA damage, and cell death (1, 2, 4, 13, 38, 39). We determined the effects of chloride substitution and Cl- channel blockers on these indexes of oxidant-induced cell injury. Oxidant-induced loss of membrane integrity to macromolecules, i.e., LDH, was detectable after 2 h of oxidant exposure and increased progressively thereafter (Fig. 1). Substitution of Cl- by large anions, e.g., isethionate, gluconate, and methanesulfonate, markedly reduced LDH release (Fig. 2). Moreover, there was a clear relationship between the extent of oxidant-induced LDH release and the concentration of Cl- in the extracellular solution (Fig. 3). These results indicate that the presence of Cl- is important for the loss of membrane integrity during oxidant injury. These results do not, however, address whether the presence of Cl- per se or Cl- entry accounts for the observed effects on LDH release. In this regard, the observation that several Cl- channel blockers reduced substantially oxidant-induced LDH release (Fig. 4), along with previous studies showing an increase in Cl- uptake during hypoxic and toxic injury (27, 31, 43), supports the view that Cl- entry mediated by Cl- channels, rather than the mere presence of Cl-, contributed to the membrane injury. Likewise, the fact that small anions, e.g., nitrate and bromide, also supported LDH release indicates that the effects on LDH release are not specific to Cl- and provides indirect support for the view that anion entry contributes to membrane injury.

The mechanism whereby Cl- or Cl- entry contributes to cell injury is not certain. The studies of Schnellmann et al. (27, 43) and our previous study of hypoxic injury in rat proximal tubules (31) were consistent with the view that Cl- entry occurred at a rather late stage in cell injury and that the effects of Cl- entry, and of Cl- channel blockers, were limited mainly to the cell membrane. The present results provide additional support for that view. Specifically, Cl- channel blockers, while reducing oxidant-induced LDH release, did not protect cells against oxidant-induced ATP depletion (Fig. 5), oxidant-induced DNA damage (Fig. 6), or oxidant-induced lipid peroxidation (Fig. 7). These observations also indicate that the Cl- channel blockers were not acting simply as antioxidants or free radical scavengers to reduce LDH release.

Chen and Mandel (10) have shown that in the course of hypoxic injury to proximal tubules, cell membranes become permeable to progressively larger molecules. That is, membranes first become permeable to small molecules and then later to macromolecules. Dong et al. (15) have also examined the size selectivity of the plasma membrane during chemical ATP depletion in MDCK cells using fluoresceinated dextrans of graded sizes. After 2 h of ATP depletion, cell membranes were freely permeable to dextran of 4 kDa, partly permeable to 70-kDa dextran, and only slightly permeable to dextrans larger than 145 kDa. Our results are also consistent with the presence of size-selective defects during oxidant-induced injury. Namely, using Cl- channel blockers or Cl- substitution (not shown), we were able to dissociate the increase in permeability to small molecules (mol wt <1,000), i.e., trypan blue (Fig. 8) or chromium (Fig. 9), from the release of the macromolecule LDH (Figs. 2-4). Chen and Mandel (10) found that cross-linking of membrane proteins with homobifunctional reagents also could reduce the anoxia-induced permeability to LDH but not to propidium iodide (mol wt = 668). Thus the effects of Cl- channel blockers in the present study were similar to the effects of cross-linking reagents in that study. It has been proposed that the permeability to macromolecules during injury results from a rearrangement of membrane proteins to form large water-filled pores (10, 15). If so, then Cl- channel blockers, either directly or indirectly, may prevent the formation of these pores. Of interest, Chen and Mandel (10) found that the permeability to small molecules was a potentially reversible event upon reoxygenation. Thus the use of small "vital" dyes, such as trypan blue, calcein, or chromium, as indicators of cell death may be misleading under certain circumstances.

The mechanism whereby Cl- substitution and Cl- channel blockers inhibit membrane permeability to macromolecules remains unknown. An increase in cell volume due to Cl- influx does not appear to be sufficient, in itself, to account for macromolecular leak. For example, cell swelling induced by osmotic gradients produced LDH release only at extremes of cell volume (11). In addition, glycine prevented LDH release from ATP-depleted MDCK cells without preventing cell swelling (15). However, a change in the intracellular Cl- content or concentration could affect steps proximal to membrane injury, e.g., by facilitating changes in the cytoskeleton (9, 14) or activation of intracellular proteases. The cellular K+ content, for example, appears to be a critical factor in the activation of proteases during apoptosis (19). The dependence of enzymes such as calpain and caspases, phospholipases, and endonucleases, all of which are activated during hypoxic injury and are proposed to contribute to cell death (16, 17, 22, 30, 40), on Cl- has not been determined. Alternatively, the effects of Cl- channel blockers could be related to their effects on the transport of substances other than Cl-. In this regard, increases in cell volume, which occur during many forms of cell injury, activate volume-sensitive anion channels (36). These channels are permeable to a variety of organic osmolytes, including glycine (37). By reducing activation of these volume-sensitive anion channels, Cl- channel blockers and Cl- substitution could prevent the efflux of potentially protective substances. The present results provide several lines of support, albeit indirect, for the view that volume-sensitive anion channels may be activated and contribute to cell injury. First, the anion dependence of H2O2-induced LDH release followed the same pattern as the selectivity sequence of volume-sensitive outward-rectifying Cl- channels (29, 36). Second, certain volume-sensitive channels require intracellular ATP for activation (5, 20), and we found that severe depletion of ATP with antimycin A markedly reduced oxidant-induced LDH release (Fig. 12). We note that, although H2O2 itself led to an approximate 60% decrease in ATP levels, the addition of antimycin A reduced ATP levels by over 95% (not shown). It is possible that some of the protection afforded by NPPB on LDH release (Fig. 4) could be related to its effect on cell ATP levels (Fig. 5). Third, volume-sensitive Cl- channels are inhibited by a number of nonclassic Cl- channel blockers, including ketoconazole and tamoxifen (25, 46). We found that these agents also markedly reduced oxidant-induced LDH release (Fig. 12). Finally, the addition of impermeant solutes (200 mM mannitol, maltose, or raffinose) to the extracellular solution to prevent cell swelling markedly reduced oxidant-induced LDH release (Fig. 11). Direct electrophysiological measurement will be required to confirm that the effects of these maneuvers are mediated through inhibition of volume-sensitive channels.

Finally, since the effects of Cl- channel blockers appeared to be limited to the plasma membrane, while oxidant-induced ATP depletion (Fig. 5), DNA damage (Fig. 6), and lipid peroxidation (Fig. 7) were unaffected, it was relevant to determine whether these agents actually provided any long-term benefit against oxidant injury. In this regard, previous studies employing freshly isolated proximal tubules (31, 43) could not address the effects of Cl- channel blockers on long-term survival due to the limited viability of tubules in suspension, and the study of Venkatachalam et al. (41) in MDCK cells examined only the end point of LDH release after 6 h of ATP depletion. Our results indicated that neither Cl- channel blockers nor glycine, an extensively studied cytoprotective agent (15, 26, 27, 41, 44, 45), enhanced cell survival after oxidant injury. These results are consistent with the view, mentioned above, that the effects of Cl- channel blockers are primarily on the plasma membrane, whereas other targets of oxidant stress are not protected. It is not known whether the failure of Cl- channel blockers to enhance survival in oxidant injury can be extrapolated to other forms of acute cell injury, such as that induced by ischemia or toxins. Further studies will be required to address that issue. However, these results should prompt caution in interpreting the effects of any maneuver, i.e., acidosis (43, 45), glycine (44), or enzyme inhibition (16, 17, 38), based solely on short-term in vitro studies or end points such as LDH release.

In summary, substitution of Cl- by impermeant anions in the extracellular solution or the addition of Cl- channel blockers prevented oxidant-induced permeabilization of the cell membrane to macromolecules. These maneuvers did not prevent oxidant-induced DNA damage, ATP depletion, lipid peroxidation, or membrane permeability to small molecules. Likewise, Cl- channel blockers did not improve cell survival, as judged by mitochondrial function 24 h following oxidant injury. These results are consistent with the view that Cl- channels may play a role in membrane damage during cell injury. Further studies are required to determine the electrophysiological features of Cl- channels in injured cells and to define the mechanism whereby Cl- channels influence membrane integrity.


    ACKNOWLEDGEMENTS

This work was supported by awards from the Extramural Grant Program of Baxter Healthcare and the Veterans Affairs Research Service. W. B. Reeves is an Established Investigator of the American Heart Association.


    FOOTNOTES

Present address of X. Meng: Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: W. B. Reeves, Division of Nephrology, Slot 501, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205 (E-mail: ReevesWilliamB{at}exchange.uams.edu).

Received 2 October 1998; accepted in final form 17 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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