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Role of Mitochondrial Na⁺ Concentration, Measured by CoroNa Red, in the Protection of Metabolically Inhibited MDCK Cells

Szilvia Baron^*,, Adrian Caplanusi^*,, Martin van de Ven^*, Mihai Radu, Sanda Despa^||, Ivo Lambrichts^¶, Marcel Ameloot^*, Paul Steels^* and Ilse Smets^*

^* Laboratory of Cell Physiology and ^¶ Laboratory of Histology, University Hasselt and transnationale Universiteit Limburg, Biomedisch Onderzoeksinstituut, Diepenbeek, Belgium; Department of Botany, Szeged University, Szeged, Hungary; Department of Medical Biochemistry, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania; Department of Health and Environmental Physics, Horia Hulubei National Institute for Physics and Nuclear Engineering, Bucharest, Romania; and ^|| Department of Physiology, Loyola University Chicago, Maywood, Illinois

Address correspondence to: Dr. Ilse Smets, MBW, Laboratory of Physiology, University Hasselt and transnationale Universiteit Limburg, Biomedisch Onderzoeksinstituut, Agoralaan Gebouw D, B-3590 Diepenbeek, Belgium. Phone: +32-11-26-85-35; Fax: +32-11-26-85-99; E-mail: ilse.smets{at}uhasselt.be

Received for publication January 19, 2005. Accepted for publication August 17, 2005.

	Abstract

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In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca²⁺. In a previous study, it was suggested that the clearance of cytosolic Ca²⁺, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na⁺/Ca²⁺ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na⁺ concentration ([Na⁺]_m) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na⁺]_m. In the first 15 min of MI, a twofold increase of [Na⁺]_m was observed reaching 113 ± 7 mM, whereas the cytosolic Na⁺ concentration ([Na⁺]_c) elevated threefold, to a level of 65 ± 6 mM. In the next 45 min of MI, [Na⁺]_m dropped to 91 ± 7 mM, whereas [Na⁺]_c further increased to 91 ± 4 mM. The striking rise in [Na⁺]_m is likely sufficient to sustain the driving force for mitochondrial Ca²⁺ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na⁺]_m was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na⁺]_m measurements in nonpermeabilized living cells.

	Introduction

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Although renal ischemia is widely examined, its underlying cellular mechanisms of ion homeostasis are not completely understood yet. One of the important determinants of ischemic injury is the cellular Ca²⁺ overload (1,2). Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca²⁺ by their fast, high-capacity, and reversible Ca²⁺ sequestration properties (3–5). The normal route for Ca²⁺ uptake into mitochondria is the Ca²⁺ uniporter, which depends on the mitochondrial membrane potential (_m) (6–8). Because metabolic inhibition (MI) induces mitochondrial depolarization (9,10), the mitochondrial Ca²⁺ uptake is highly diminished via the Ca²⁺ uniporter (11,12). Because the stoichiometry of the mitochondrial Na⁺/Ca²⁺ exchanger (NCX) might be closer to 3Na⁺/1Ca²⁺ than 2Na⁺/1Ca²⁺ (8,13), the pronounced mitochondrial depolarization during MI strongly diminishes the electrical driving force for Ca²⁺ efflux from the mitochondria via the NCX and might allow the reverse action of the mitochondrial NCX. Moreover, it was reported that the mitochondrial NCX might reverse during MI in rat cardiomyocytes (14). In our previous study (9), the mitochondrial Ca²⁺ uptake was shown to be Na⁺ dependent, and the clearance of cytosolic Ca²⁺ overload was suggested to occur via the reverse action of the mitochondrial NCX. We hypothesized that the cytosolic Ca²⁺ overload (from approximately 50 nM to approximately 630 nM), induced a chemical driving force for Ca²⁺ entry into the mitochondrial matrix. Therefore, in the initial phase of the cytosolic Ca²⁺ clearance, the reversed mode of the mitochondrial NCX is likely driven by the Ca²⁺ gradient toward the mitochondrial matrix. However, when the Ca²⁺ gradient gradually decreases, a significant increase of mitochondrial Na⁺ concentration ([Na⁺]_m) might sustain the driving force for the reverse action of mitochondrial NCX. It is known that some renal tubular cells, predominantly cortical ascending limb cells, collecting duct cells, and cells of the tubules within the inner medulla, seem to escape ischemic injury or are only sublethally injured and are capable of complete functional and structural recovery if the insult is removed in time (15).

The objective of this study was to corroborate our previous suggestion that the mitochondrial NCX reverses during MI. Therefore, changes in [Na⁺]_m were monitored in metabolically inhibited MDCK cells, a cell line of distal tubular origin that possesses many similarities with mammalian cortical collecting tubular cells (16). Inhibition of cellular metabolism was used as an in vitro experimental model for ischemic cell injury (17). MI was induced by inhibiting both cellular glycolysis (with 2-deoxyglucose) and oxidative phosphorylation (with cyanide). Furthermore, to investigate whether the mitochondria might protect the cell during MI from injury by the reversal of the mitochondrial NCX, we monitored the cellular ATP level ([ATP]_c), _m, and the cytosolic Na⁺ concentration ([Na⁺]_c) of MDCK cells during recovery.

	Materials and Methods

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Solutions and Chemicals
MDCK cells were bathed in a normal saline solution (NSS) that contained (in mM) 140 NaCl, 5 KCl, 1.5 CaCl₂, 1 MgSO₄, 10 HEPES, and 5.5 glucose (pH adjusted to 7.4 with TRIS). MI was accomplished with a solution that contained (in mM) 135 NaCl, 5 KCl, 1.5 CaCl₂ 1 MgSO₄, 10 HEPES, 10 2-deoxy-d-glucose (2-DG), and 2.5 NaCN (pH 7.4). Recovery (reperfusion) was carried out with NSS. CoroNa Red, Mito Tracker Green (MTG), and pluronic F-127 were obtained from Molecular Probes, Inc. (Eugene, OR). Gramicidin D, carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), and nigericin were bought from Sigma (St. Louis, MO). CGP-37157 monensin and oligomycin were purchased from Tocris (Bristol, UK). All chemicals used were of analytical grade.

Cell Culture
MDCK cells (low passage number 22 to 30) were donated by Dr. H. De Smedt (Laboratory of Physiology, Leuven, Belgium). Cells were cultured in a 1:1 mixture of DMEM and Ham’s F-12 (N.V. Invitrogen, Carlsbad, CA) supplemented with 10% FCS, 14 mM l-glutamine, 25 mM NaHCO₃, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained in a humidified 5% CO₂ atmosphere at 37°C. The medium was renewed every 3 to 4 d. For all experiments, 0.5 to 1 x 10⁵ cells were seeded on round glass coverslips with a diameter of 24 mm. After 3 to 6 d, confluent monolayers were used.

Cellular ATP Content
ATP measurements were performed with a luciferin-luciferase–based assay kit (Molecular Probes) (9). ATP levels were measured with a luminometer model 1250 from Wallac (Turku, Finland).

Fluorescence Imaging Microscopy
An Axiovert 100 inverted epifluorescence microscope (Carl Zeiss, Jena, Germany) was used. Fluorescence was elicited by a XBO 75 W/2 OFR xenon lamp (Osram, Berlin-München, Germany). A Zeiss objective LD Achroplan 40x/0.6 corr was used for all images. The images were captured by a Quantix CCD camera (Photometrics, Tucson, AZ). All optical filters and dichroic mirrors were obtained from Chroma Technology Corp. (Brattleboro, VT). Cells were grown on glass coverslips, placed into a homemade holder, and put on the microscope stage.

Determination of _m.
_m was evaluated using the potentiometric indicator 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1) (9). The fluorescence of JC-1 was monitored at 535 nm (F₅₃₅) and at 590 nm (F₅₉₀). The F₅₃₅ signal, corresponding to the JC-1 monomer, is responsive to values of |_m| <140 mV (10). In the underlying study, the F₅₃₅ signal is primarily mitochondrial because analysis of the F₅₃₅ JC-1 image only at mitochondrial positions (determined by the positions of aggregate fluorescence in the paired F₅₉₀ image) gives rise to a nearly identical behavior of the resulting mitochondrial F₅₃₅ signal during MI as compared with the "unmasked" F₅₃₅ signal (data not shown). The 590-nm emission, corresponding to the JC-1 aggregate, shows very little sensitivity in the range of |_m| <140 mV, but aggregate fluorescence is sensitive to a drop of |_m| from approximately 200 to approximately 140 mV (10). Because both JC-1 signals are changing during metabolic inhibition in MDCK cells (data not shown) and the F₅₃₅ signal is primarily mitochondrial, the authors take both fluorescence signals into account to obtain an accurate measure of _m at both the high and low potential range. For comparing experiments with different control values of the JC-1 emission ratio, R (F₅₉₀/F₅₃₅), results are presented in terms of a normalized ratio (R_norm):

where R_control and R_FCCP are the emission ratios under control conditions and after adding FCCP plus 2-DG, respectively.

Determination of Cytosolic Na⁺ and Ca²⁺ Concentrations.
Cytosolic Na⁺ and Ca²⁺ concentrations ([Na⁺]_c and [Ca²⁺]_c) were monitored using, respectively, the fluorescence probes SBFI and Fura-2 as described previously (9). The autofluorescence intensity, measured at the excitation wavelengths (340 and 380 nm) and the emission wavelength 535 nm (the wavelengths used in Fura-2 and SBFI experiments), did not change during metabolic inhibition in MDCK cells at 37°C (data not shown). The autofluorescence image of unloaded cells was subtracted from the loaded cell images.

Laser Scanning Confocal Microscopy
A Zeiss LSM 510 META laser scanning confocal microscope (Carl Zeiss) attached to an Axiovert 200 M frame (Carl Zeiss) was used. The microscope stage is equipped with a model P heated specimen holder with type S incubator (PeCon GmbH, Erbach-Bach, Germany). Fluorescence measurements were performed with a 63X/1.4 Plan Apochromat oil immersion objective (Carl Zeiss). For minimizing temperature differences between sample and objective, a x63 oil model objective heater (PeCon GmbH) was used. Cells were grown on glass coverslips, mounted into a homemade holder, and placed on the microscope stage.

Determination of [Na⁺]_m.
Cells were loaded with two fluorescence probes: MTG, a fluorescence probe that selectively stains mitochondria, and CoroNa Red, a sodium-sensitive probe. MDCK cells were incubated simultaneously with 200 nM MTG and 2 µM CoroNa Red in 1 ml of NSS that contained 0.025 wt/vol % pluronic F-127 for 30 min at 37°C. CoroNa Red is a cationic dye that can be loaded into the cell without acetoxymethyl ester groups. Because CoroNa Red has a positive charge, it is taken up preferentially into polarized mitochondria, where it reports changes in [Na⁺]_m by changes in its fluorescence intensity. After loading, cells were washed gently twice with NSS. MTG was excited by an Ar laser (488 nm), with a power of 0.1 mW at the sample position. The excitation of CoroNa Red was carried out with a Green HeNe laser (543 nm), with a power of 0.01 mW at the sample position. Fluorescence emission of MTG and CoroNa Red was collected via Neben Farb Teiler 490 and Neben Farb Teiler 545 dichroic mirrors and 525/25-nm band-pass and 560-nm long-pass filters, respectively. Each image was collected with 512 x 512 pixels. The pixel dwell time was 26.5 µs. The pinhole was 3 Airy units in all experiments. All images were collected with a digital zoom factor of 1.

The CoroNa Red fluorescence had to be corrected for nonmitochondrial CoroNa Red fluorescence, including the weak cytosolic CoroNa Red staining and the pronounced staining of structures within the nuclei, presumably nucleoli. Within the histogram of the MTG image, a threshold was chosen to retain only the high-end tail, excluding any saturated pixels (9). Resulting MTG intensities represented upon visual inspection the "well-delineated" mitochondrial locations. The intensities of the selected mitochondrial pixels were set at 1, whereas all other pixel intensities in the MTG image were set at 0. Multiplication of this "mask" image with the CoroNa Red image yielded an image that consisted of mitochondrial CoroNa Red fluorescence intensity. This value was normalized with regard to the number of pixels with mask value 1 in the "mask" images to compensate for differences in mitochondrial density. Furthermore, because the mitochondria moved continuously in the cytosol, both an MTG image and a CoroNa Red image were collected at each time point. For image transfer, conversion, and processing, the Zeiss LSM Image Browser and ImageJ Java–based freeware (Research Services Branch, National Institute of Mental Health/National Institute of Neurological Disorders and Stroke, Bethesda, MD) plugin routines were used.

In vivo calibration of the fluorescence signal of CoroNa Red in MDCK cells was accomplished by exposing the cells to various extracellular Na⁺ concentrations in the presence of gramicidin D (10 µM, from 2 mM stock solution in ethanol) to equilibrate the Na⁺ gradient across the plasma membrane, nigericin (10 µM, from 13 mM stock solution in ethanol) to equilibrate the pH gradient across the plasma membrane, monensin to equilibrate Na⁺ gradients across the mitochondrial membrane (20 µM, from 10 mM stock in ethanol), FCCP (1 µM, from 1 mM stock solution in DMSO) to equilibrate pH gradients across the mitochondrial membrane, and additionally oligomycin (20 µg/ml, from 5 mg/ml stock in ethanol) to inhibit the mitochondrial ATPase. The solutions with various sodium concentrations were prepared by mixing in different proportions two solutions of equal ionic strength and osmolality. One solution contained 145 mM Na⁺ (30 mM NaCl and 115 mM Na gluconate) and no K⁺, whereas the other one included 145 mM K⁺ (30 mM KCl and 115 mM K gluconate) and was Na⁺ free. Both calibration solutions also contained 1.5 mM CaCl₂, 1 mM MgSO₄, 10 mM HEPES, and 5.5 mM glucose. The pH was adjusted to 7.4 with 0.1 M TRIS. The sodium concentration of the solution was set to five different values in the range of 0 to 145 mM. The plot of the normalized fluorescence intensity of CoroNa Red against [Na⁺]_m resulted in a logarithmic curve (Figure 1).

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. In vivo calibration curve of the mitochondrial CoroNa Red signal in MDCK cells. Mitochondrial Na+ concentration ([Na+]m) as a function of the fluorescence intensity of the normalized CoroNa Red signal (n = 7) ± SE. The control CoroNa Red fluorescence intensity is defined as the fluorescence intensity in normal, untreated MDCK cells after loading with CoroNa Red.

Cell Volume Measurements
This method has been described previously in detail (18). Cell thickness (T_c) was used as an index for cell volume of confluent monolayers. The apical (upper) side of the monolayer was labeled with fluorescence biotin-coated microbeads. Focusing of the microbeads was automatically performed with a piezoelectric focusing device (PIFOC; Physik Instrumente, Waldbronn, Germany). T_c is defined as the vertical distance between the basolateral and apical beads. Measured T_c values were corrected for the diameter of the fluorescence microbeads by subtracting 1 µm. Changes in cell height are expressed as percentage of the value recorded just before MI is imposed. Averaged values of T_c were calculated using the recordings from a number of beads (n_B) that remained attached to the monolayer during the entire experiment.

Statistical Analyses
Values from N different monolayers are given as means ± SEM. All experiments described in this article were performed at 37°C.

	Results

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Imaging Mitochondrial Sodium with Confocal Microscopy
To evaluate changes in [Na⁺]_m during MI, we performed experiments on MDCK cells that were loaded with both the mitochondrion-specific dye MTG and the Na⁺-sensitive probe CoroNa Red. Under control conditions, the mitochondria seem to be organized in wire-like structures (Figure 2A). Incubation with metabolic inhibitors induced breakage of the mitochondrial network (Figure 2B). During MI, MDCK cells swell rapidly (Figure 2H). This cellular reorganization induces an increase in the contribution of mitochondria above and below the image plane, resulting in fuzzy confocal images during MI. The changes in mitochondrial shape are related to MI rather than resulting from a time effect, because incubation in NSS still revealed wire-like mitochondria after a 60-min incubation period (images not shown). The mitochondrial network was partially rebuilt in metabolically inhibited cells after a 30-min recovery period in NSS without metabolic inhibitors (Figure 2C). The superimposed images of CoroNa Red and MTG (Figure 2, D through F) show that CoroNa Red is located mainly in mitochondria when loaded in MDCK cells. Nevertheless, a weak staining of the cytosol and a pronounced staining of structures within the nuclei of the cells, presumably nucleoli, occurred. To resolve the mitochondrial CoroNa Red fluorescence from nonmitochondrial CoroNa Red contributions, we used a "mask procedure" based on mitochondrial localization in the MTG image (for details, see Experimental Procedures section). The resulting "mitochondrial" CoroNa Red image (Figure 2G) confirms the spatial correlation of MTG and CoroNa Red in mitochondria, because only CoroNa Red intensities that correspond with MTG-positive pixels in the MTG mask are retained.

View larger version (51K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Confocal images of MDCK cells that were loaded with Mito Tracker Green (MTG) and CoroNa Red. The mitochondrial structure in control conditions (A), after 60 min of metabolic inhibition (MI; B), and after a subsequent 30-min recovery phase (C). MTG (D) and CoroNa Red (E) staining of mitochondria in cells after a 30-min incubation period with metabolic inhibitors. The staining with the Na+-sensitive probe CoroNa Red was spatially correlated with the mitochondrial marker MTG (F), except for a weak CoroNa Red staining of the cytosol and a more pronounced staining of nuclear structures, presumably nucleoli (arrows). Application of the "mask" procedure (for details, see Materials and Methods section) yielded an image that consisted of only mitochondrial CoroNa Red fluorescence intensities (G; scale bar = 10 µm). Effect of MI on cell thickness (Tc) in MDCK cells (H). Shown are the mean (solid line) and the SEM (dotted lines) from four different monolayers (nB = 57). The initial absolute Tc value was 10.97 ± 0.90 µm.

Effect of _m on the Retention of CoroNa Red

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. The effect of mitochondrial membrane potential (m) dissipation on the mitochondrial entrapment of CoroNa Red in MDCK cells. For abolishing the m, MDCK cells were exposed to 1 µM carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP). CoroNa Red remained in the mitochondria independent of the dissipated m. [Na+]m values are indicated as means (n = 5) ± SE.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Changes in cytosolic and mitochondrial Na+ concentrations during MI in MDCK cells. (A) In metabolically inhibited MDCK cells, a striking increase in [Na+]m was seen followed by a slight decrease (; n = 7), whereas in control tissues, incubated in normal saline solution (NSS), no significant changes of [Na+]m were observed (•; n = 3). (B) Inhibition of cellular metabolism induced a steady increase in [Na+]c (; n = 7) and a striking increase in [Na+]m followed by a slight decrease (; n = 7). Values are indicated as means ± SEM.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Influence of CGP-37157 on [Na+]m changes during MI in MDCK cells. CGP-37157 (25 µM) was added during a 30-min preincubation period and during a subsequent 60-min incubation period simultaneous with metabolic inhibitors. [Na+]m values are indicated as means (n = 3) ± SE.

Changes in MDCK Cells during Recovery Phase after 60 Min of MI
To investigate whether MDCK cells can recover after 60 min of MI, we placed the cells in NSS (recovery) and monitored changes in [ATP]_c, _m, and [Na]_c (Figure 6). The cellular ATP content increased to approximately 50% of the control level in the first 5 min of recovery. After 30 min, [ATP]_c was approximately 70% of the control level (Figure 6A). _m increased to approximately 70% of the control level in the first 3 min of the recovery phase. However, this value did not increase further in the next 27 min (Figure 6B). [Na]_c started to recover after NSS was applied and decreased from approximately 90 mM to approximately 60 mM in 30 min (Figure 6C).

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Recovery of MDCK cells after 60 min of MI. After 60 min of MI, MDCK cells were placed in NSS. Changes in the cellular ATP content (n = 4; A), m (n = 8; B), and [Na+]c (n = 7; C) are shown. ATP levels are indicated as percentage of the level in control conditions. Values are indicated as means ± SE.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Recovery after MI requires the activity of the mitochondrial Na+/Ca2+ exchanger (NCX) during MI. Metabolically inhibited MDCK cells were treated with CGP-37157 (25 µM) before and during MI. The cells were bathed in NSS without metabolic inhibitors and without CGP-37157 in the recovery phase. (A) m, assessed by the ratio Rnorm, and (B) intracellular calcium concentrations ([Ca2+]c) were monitored with Fura-2. Each panel shows the mean values ± SE from three monolayers.

	Discussion

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In this study, changes in [Na⁺]_m were monitored in metabolically inhibited MDCK cells, using the fluorescent dye CoroNa Red, to verify our hypothesis that the clearance of cytosolic Ca²⁺ overload during MI in renal epithelial cells occurs via the reverse action of the mitochondrial NCX. Furthermore, the ability of MDCK cells to recover from a 60-min period of MI was examined.

Mitochondrial Na⁺ Content in MDCK Cells
Studies in isolated mitochondria reported that [Na⁺]_m is lower than [Na⁺]_c (23). A study on permeabilized myocytes suggested that [Na⁺]_m is approximately half of [Na⁺]_c (24). Electron probe analysis on dry cryosections of vascular smooth muscle showed nearly equal cytosolic and [Na⁺]_m (25). There is no information available concerning [Na⁺]_m in living cells. In our experiments, [Na⁺]_m was monitored noninvasively in living MDCK cells. Under control conditions, [Na⁺]_m was approximately 50 mM, a two times higher value than [Na⁺]_c (Figure 4B). The observed Na⁺ gradient across the mitochondrial membrane is likely maintained by the electrogenic mitochondrial NCX (13) and the highly negative _m. When CGP-37157, a specific inhibitor of the mitochondrial NCX, was administered during 30 min under control conditions, [Na⁺]_m decreased to approximately 25 mM (Figure 5), a similar value as [Na⁺]_c. This result supports the assumption that the mitochondrial NCX maintains the high mitochondrial sodium level. In the cell, the priority is likely to keep the mitochondrial Ca²⁺ level ([Ca²⁺]_m) low, enabling the mitochondria to buffer rapidly any cytosolic Ca²⁺ overloads.

Na⁺ Content in Nonrespiring Mitochondria of MDCK Cells
The addition of the protonophore FCCP diminishes _m and the H⁺ gradient across the mitochondrial membrane (26,27). The reduction of the H⁺ gradient likely strongly decreases the rate of the mitochondrial Na⁺/H⁺ exchanger (NHE), the normal route for Na⁺ efflux. However, the mitochondrial Na⁺ uptake via the NCX is functional. The blockage in the Na⁺ efflux elicits a significant increase in [Na⁺]_m (Figure 3).

Changes in Mitochondrial Na⁺ in MDCK Cells during MI
During MI, [Na⁺]_m showed a similar behavior (Figure 4A) as in the case of applying FCCP (Figure 3). Consequently, the underlying mechanism might be analogous. Because _m significantly decreases during MI (9), one can speculate that the H⁺ gradient across the mitochondrial membrane might decrease as well. Therefore, the initial increase of [Na⁺]_m during MI might be due to the restricted mitochondrial Na⁺ efflux via the arrested NHE activity, whereas the mitochondrial Na⁺ uptake is still active via the mitochondrial NCX. After 15 min of MI, the pronounced depolarization of _m and a high Ca²⁺ gradient toward the mitochondrial matrix (9) favors the reversal of the mitochondrial NCX. Therefore, the second-phase decrease of [Na⁺]_m is likely due to the Na⁺ efflux via the reversed mitochondrial NCX. This is supported by the result obtained in the presence of CGP-37157, a specific inhibitor of the mitochondrial NCX, because the second-phase drop of [Na⁺]_m was completely abolished.

The time course of [Na⁺]_m increase in response to MI is different in CGP-37157–treated MDCK cells. At the moment MI is applied, the conditions are different for the experiments in Figures 4B and 5. When CGP-37157 is added, [Na⁺]_m drops immediately to a value similar to the cytosolic concentration. We assume that [Na⁺]_m in that condition will be determined mainly by the mitochondrial NHE. When MI is applied, it induces an increase in [Na⁺]_c. Moreover, an acidification in both the mitochondrial matrix and the cytosol can be expected (28). The rate of mitochondrial sodium uptake will be determined by two driving forces: The sodium gradient toward the mitochondrial matrix and the H⁺ gradient across the inner mitochondrial membrane, which is unknown. This H⁺ gradient is not expected to be large, because both compartments (the mitochondrial matrix and the cytosol) will acidify as a result of the hydrolysis of ATP. Therefore, the increase in [Na⁺]_m follows the increase in [Na⁺]_c, which saturates at approximately 45 min. The reason that [Na⁺]_m is higher than [Na⁺]_c is probably because the mitochondrial matrix acidified to a greater extent. In the absence of the blocker CGP-37157 (Figure 4B), the transport of Na⁺ across the inner mitochondrial membrane is determined by two exchangers: The NHE (electroneutral) and the NCX (electrogenic). Hence, the gradients of Ca²⁺, Na⁺, and H⁺ and the mitochondrial membrane potential will influence mitochondrial Na⁺ handling. Because in the experiments of Figure 4B the normal Na⁺ influx pathway in the mitochondria is functionally active, Na⁺ will enter the mitochondrial matrix in the initial phase of MI via the NCX. To conclude, Figures 4B and 5 show [Na⁺]_m changes during MI in clearly different conditions.

Driving Forces for the Reverse Action of the Mitochondrial Na⁺/Ca²⁺ Exchanger during MI
Our previous study (9) showed that the mitochondria play a key role in the clearance of cytosolic Ca²⁺ during MI in MDCK cells. Because |_m| was largely diminished after 20 min of MI, the normal route for mitochondrial Ca²⁺ uptake via the _m-dependent Ca²⁺ uniporter was limited. However, the depolarization of the mitochondria strongly reduces the electrical driving force for Ca²⁺ efflux via the putative electrogenic NCX (7,13), and an approximately 12-fold increase of [Ca²⁺]_c after 18 min of MI induces a chemical driving force for Ca²⁺ entry into the mitochondrial matrix. These changes in electrochemical gradients might allow a reverse action of the mitochondrial NCX. In this study, after 15 min of MI, an approximately 2.2-fold increase of [Na⁺]_m was detected. Because after 15 min of MI _m is strongly dissipated, the sodium gradient (Figure 4B) promotes the reversal of the mitochondrial NCX. The peak in [Ca²⁺]_c (9) occurred 3 min after the peak in [Na⁺]_m. The reversed mitochondrial NCX only then becomes efficient in clearing the entered Ca²⁺ from the cytosol.

Possible Protection Mechanism of MDCK Cells from Lethal Injury by Reversed Action of Mitochondrial NCX
After 30 min of recovery from 60 min of MI, the cytosolic [ATP]_c and _m were largely restored, whereas [Na⁺]_c decreased by approximately 30% (Figure 6). The complete recovery probably requires a significantly longer period. The absence of _m recovery in CGP-37157–treated metabolically inhibited MDCK cells suggests that a functioning mitochondrial NCX during MI is necessary to enable recovery after washout of the metabolic inhibitors. Furthermore, normal cytosolic calcium concentrations were not restored in CGP-37157–treated MDCK cells after MI. Our results suggest that both mitochondrial and cellular recovery depends on the activity of the mitochondrial NCX during MI.

In summary, the results of this study confirm our previous suggestion that the mitochondrial NCX reverses during MI to carry out the clearance of the cytosolic Ca²⁺ in MDCK cells. The reversal of the mitochondrial NCX during MI seems to play an important role in the protection of the cells, because NCX inhibition prevents both mitochondrial and cellular recovery.

	Acknowledgments

 
This work was supported by a bilateral research collaboration program between Flanders and Hungary (BIL01/18HON), a bilateral research collaboration program between Flanders and Romania (BIL00/40ROE), and by the tUL impulsfinanciering.

We acknowledge J. Janssen for excellent technical assistance with the cell culture. We thank R. Beenaerts, W. Leyssens, P. Pirotte, M. Jans, and R. Van Werde for technical help. We acknowledge Dr. W. Van Driessche for help with the cell volume measurements. We are grateful to various groups for the free software for confocal image analysis: LSM Reader plugin (University of Strasbourg, Strasbourg, France; Dr. J. Mutterer, Dr. Y. Krempp, and Dr. P. Pirrotte), ImageJ (NIH, Bethesda, MD; Dr. W. Rasband and available online: http://rsb.info.nih.gov/ij/plugins/lsm-reader.html), and VolumeJ (Utrecht University, Utrecht, The Netherlands; Dr. M. Abramoff).

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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