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Am J Physiol Renal Physiol 293: F533-F540, 2007. First published May 23, 2007; doi:10.1152/ajprenal.00030.2007
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Metabolic inhibition-induced transient Ca2+ increase depends on mitochondria in a human proximal renal cell line

Adrian Caplanusi,1,3 Andrew J. Fuller,1 Romer A. Gonzalez-Villalobos,1 Timothy G. Hammond,1,2,4 and L. G. Navar1

1Department of Physiology and Hypertension and Renal Center of Excellence and 2Nephrology Section, Department of Internal Medicine, Tulane University Health Sciences Center, New Orleans; 3Department of Medical Biochemistry, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania; and 4Southeast Louisiana Veterans Healthcare System, New Orleans, Louisiana

Submitted 16 January 2007 ; accepted in final form 19 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
During ischemia or hypoxia an increase in intracellular cytosolic Ca2+ induces deleterious events but is also implicated in signaling processes triggered in such conditions. In MDCK cells (distal tubular origin), it was shown that mitochondria confer protection during metabolic inhibition (MI), by buffering the Ca2+ overload via mitochondrial Na+-Ca2+ exchanger (NCX). To further assess this process in cells of human origin, human cortical renal epithelial cells (proximal tubular origin) were subjected to MI and changes in cytosolic Ca2+ ([Ca2+]i), Na+, and ATP concentrations were monitored. MI was accomplished with both antimycin A and 2-deoxyglucose and induced a 3.5-fold increase in [Ca2+]i, reaching 136.5 ± 15.8 nM in the first 3.45 min. Subsequently [Ca2+]i dropped and stabilized to 62.7 ± 7.3 nM by 30 min. The first phase of the transient increase was La3+ sensitive, not influenced by diltiazem, and abolished when mitochondria were deenergized with the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The subsequent recovery phase was impaired in a Na+-free medium and weakened when the mitochondrial NCX was blocked with 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Thus Ca2+ entry is likely mediated by store-operated Ca2+ channels and depends on energized mitochondria, whereas [Ca2+]i recovery relied partially on the activity of mitochondrial NCX. These results indicate a possible mitochondrial-mediated signaling process triggered by MI, support the hypothesis that mitochondrial NCX has an important role in the Ca2+ clearance, and overall suggest that mitochondria play a preponderant role in the regulation of responses to MI in human renal epithelial cells.

Ca2+ influx; energized mitochondria; mitochondrial Na+-Ca2+ exchanger; thapsigargin; CGP-37157

INTRACELLULAR Ca2+ concentration ([Ca2+]i) homeostasis depends on the activity of many cellular components including the Ca-ATPase located in the plasma membrane and in the endoplasmic reticulum (ER) membranes. Following ischemia, the ATP level drops rapidly and the efficiency of ATP-fueled Ca2+ pumps to clear Ca2+ is reduced, leading to increases in [Ca2+]i. Hence the Ca2+ overload elicited by hypoxia is partially due to the gradual reduction in ATP levels (4, 28, 39). However, there is evidence that the [Ca2+]i increase during acute hypoxic injury or metabolic inhibition (MI) is implicated in complex signaling processes (55). In regard to the kidney, the renal proximal epithelium is one of the main targets of ischemia (27), and Ca2+ overload is considered to have a major role in mediating ischemic damage at the renal cellular level (44). Therefore, the understanding of the basic mechanisms of Ca2+ dynamics is essential in designing protective strategies against hypoxic-ischemic injury.

The ability to recover after an ischemic insult is associated with many processes, including prompt reductions in the ischemia-induced [Ca2+]i increase (35, 40). In renal epithelial cells, restoring the low [Ca2+]i seems to be an important factor that increases the resistance against MI (2, 7, 46). Mitochondria are able to buffer substantial quantities of Ca2+ under physiological and pathophysiological conditions and have an important role in maintaining intracellular Ca2+ homeostasis (12, 37). Classically, the Ca2+ buffering capacity of mitochondria relies on the mitochondrial Ca2+ uniporter, whose activity is triggered by a rise in [Ca2+]i and depends on the high inner mitochondrial membrane potential ({Delta}{Psi}). However, in particular physiological conditions, when {Delta}{Psi} is reduced or collapsed (i.e., during MI), other structures could be involved in mitochondrial Ca2+ buffering. Recent reports show that mitochondria protect the renal epithelial cell against Ca2+ overload during MI by the aim of mitochondrial Na+-Ca2+ exchanger (NCX) (2, 46). These studies were carried out in MDCK cells and demonstrated that MI induces a transient increase in [Ca2+]i whose recovery phase is mediated by the mitochondrial NCX working in the entry mode. The driving force of Ca2+ entry into mitochondria via NCX is likely sustained by an MI-induced increase in intracellular Na+ concentration ([Na+]i). The reversal of mitochondrial NCX during hypoxia was reported also in rat cardiomyocytes (16, 17). However, the responses in proximal tubular cells, which are thought to have much higher metabolic activity and therefore are more vulnerable to ischemic injury, have not been evaluated previously.

The aims of the present work were to investigate whether metabolically inhibited human renal proximal tubular cells exhibit a similar transient increase in [Ca2+]i and to determine the involvement of mitochondria in the MI-induced processes. Human cortical renal epithelial cells (HCRE) (3, 9, 25), which possess the characteristic brush border markers of the proximal tubule and have been used in the past as a model for this segment, were selected. Inhibitors of ATP production were utilized because they have been used extensively to study ischemic injury in polarized epithelial cells (2, 11, 28, 46). MI, which mimics the ischemic process, was induced with both antimycin A-a blocker of complex III of mitochondrial electron transport chain (45) and 2-deoxyglucose, which blocks the glycolytic pathway in a glucose-free medium (48). To test the role of mitochondria in the [Ca2+]i time course during MI, two compounds were used: 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157), a benzodiazepine compound widely used as a specific blocker of mitochondrial NCX (8, 10, 32), and the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), which collapses the inner mitochondrial membrane potential.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells. SV-40 immortalized HCRE cells (primary cells obtained from Clonetics prior to immortalization) were used in this study. HCRE cells were previously characterized and used as a model of renal proximal epithelial cells (3, 9, 25). They show proximal tubular features including proximal tubular enzyme markers ({gamma}-glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase), form a polarized monolayer with apical microvilli, and express the proximal tubular receptors cubulin and megalin (25). They maintain active sodium-dependent transport of phosphate and glucose (3). The cells were cultured in conventional T75 flasks in an incubator with humidified atmosphere and 5% CO2, by using DMEM/F12 (GIBCO/Invitrogen, Carlsbad, CA) supplemented with heat-inactivated fetal calf serum (10%) and an antibiotic cocktail: Ciprofloxacin (Bayer, West Haven, CT) and Fungizone (GIBCO/Invitrogen). The culture medium was renewed every 48 h and a new passage was initiated when the cell monolayer became confluent, according to a standard trypsinization protocol. The cells (passages 4–26) were seeded on glass coverslips (50,000–100,000 per coverslip) and cultured for 2–3 days before the experiments were run. All experiments described in this paper were performed at 37°C.

Fluorescence imaging microscopy. The coverslips containing the HRCE cells were mounted on a 200-µl chamber (Warner Instruments, Eugene, OR) and placed on the stage of an inverted epifluorescence microscope (Nikon Diaphot, Nikon Instruments, Tokyo, Japan). The temperature of the sample was set and maintained at 37°C with the help of an electric stage heater (Warner Instruments). Cells were gently washed several times with normal saline solution, before and after dye loading. The experiments were performed with a dual monochromator-based fluorescence spectrophotometer equipped with a 75-W xenon lamp and chopper wheel (Photon Technology International, South Brunswick, NJ) (13). For both Fura-2 and sodium-binding benzofuran isophthalate (SBFI), the excitation wavelengths were set at 340 and 380 nm and the emission signal was collected at 510 nm. Slit widths of 3 nm were set for both excitation monochromators. The excitation light was directed to the sample by a dichroic mirror (DM400; Nikon Instruments) and a 40x objective (Nikon Fluor 40, oil immersion, NA = 1.3; Nikon Instruments, Tokyo, Japan). Measurements of fluorescence intensity were collected every second and were analyzed with the aid of the Photon Technology International software. The background fluorescence was automatically subtracted from the loaded cell images. The photobleaching of the fluorescent dyes (Fura-2 and SBFI) was analyzed and found to be insignificant during the time interval of the experiments performed.

Calcium measurements. [Ca2+]i was monitored by using the fluorescent probe Fura-2. The cells were incubated with the membrane-permeant acetoxymethyl (AM) ester form of Fura-2 (2 µM) for 30 min at 37°C in the presence of 0.05% wt/vol Pluronic F-127. The fluorescent signal was calibrated in vivo at the end of each experiment by the method described by Grynkiewicz et al. (18). The dissociation constant of Fura-2 for Ca2+ was considered 135 nM (49). Maximum ratio (Rmax) was obtained by permeabilizing the cells with the Ca2+ ionophore, ionomycin (10 µM), in the presence of 1.5 mM extracellular Ca2+. Subsequently, to obtain the minimum ratio Rmin, the cells were exposed to a Ca2+-free solution (containing 10 mM EGTA) with 10 µM ionomycin and 10 µM BAPTA-AM.

Sodium measurements. [Na+]i was monitored using the fluorescent probe SBFI. The cells were loaded with 2 µM SBFI-AM for 1 h at 37°C, in the presence of 0.05% wt/vol Pluronic F-127. SBFI was used in a dual-excitation ratiometric mode. The calibration of the SBFI fluorescence signals was achieved by exposing the cells to various extracellular Na+ concentrations in the presence of the ionophore gramicidin D (10 µM) at the end of each experiment. A calibration curve was derived according to the procedure described by Zahler et al. (56).

Cellular ATP content. Fully confluent HCRE monolayers were used for the determination of the ATP content. The coverslips were gently washed several times with normal saline solution and afterward subjected to MI for various time intervals ranging from 5 to 30 min. ATP measurements were performed with a luciferin-luciferase-based assay kit (Sigma, St. Louis, MO). The reaction buffer contained 150 µg/ml luciferin, 1.25 µg/ml luciferase, 5 mM MgSO4, 1 mM dithiothreitol, 25 mM tricine, 0.1 mM EDTA, and 0.1 mM azide, at pH 7.8. The cells were solubilized in 450 µl of Somatic cell ATP-releasing agent (Sigma) for 30 s, and 50 µl of cell extract was added to 450 µl of reaction buffer. ATP levels were measured with a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). The results are expressed as percent change compared with control. The control ATP values were given by monolayers (2 coverslips per point) processed in the same way but not subjected to MI.

Experimental protocols. MI was accomplished by inhibiting simultaneously the mitochondrial respiration and the glycolytic pathway by using both antimycin A and 2-deoxyglucose, respectively. The MI effects in terms of [Ca2+]i, [Na+]i, and ATP concentration were investigated during 30-min exposures. [Ca2+]i and ATP concentration were further monitored for a 10-min washout period, when the metabolic inhibitors containing solution were replaced by a normal saline solution, which was followed by another 15 min of MI. The MI effects on [Ca2+]i either in the presence of various compounds or in a Ca2+-free medium as well as a Na+-free medium were investigated during 30-min exposures.

Solutions and chemicals. HCRE cells were bathed in a normal saline solution containing (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 containing (in mM) 135 NaCl, 5 KCl, 1.5 CaCl2, 1 MgSO4, 10 HEPES, and 10 2-deoxyglucose (pH 7.4 with Tris); 10 µM antimycin A was added to the solution before use. Ca2+-free solutions contained 10 mM EGTA. N-methyl-D-glucamine replaced Na+ in the Na+-free solutions. Fura 2-AM, SBFI-AM, BAPTA-AM, and Pluronic F-127 were obtained from Invitrogen-Molecular Probes (Eugene, OR). FCCP, ionomycin, thapsigargin, gramicidin D, EGTA, and LaCl3 were provided by Sigma. CGP-37157 was obtained from Tocris (Bristol, UK).

Statistics. Data values from n different monolayers are given as means ± SE. Statistical comparisons were made with the Mann-Whitney U-test when applicable. A value of P < 0.01 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
MI was achieved by using simultaneously both metabolic inhibitors: antimycin A and 2-deoxyglucose. This treatment induces in HCRE cells an important energetic deficit. As depicted in Fig. 1, as a consequence of MI, the ATP levels dropped to 35.6% from the control in 5 min, to 13.1% in 10 min, and to 1.5% of control after 30 min of MI. On washout, the ATP level recovered after 10 min to ~17% of the control and during the second exposure to MI, the ATP level dropped to 0.9% from the control. However, we verified that the cells were alive at the end of the 30-min MI period by their capacity to recover and by the Trypan blue exclusion test (which showed >98% viability after 30 min of MI). Moreover, the cells did not detach spontaneously from the monolayer after 30 min of MI, which is also a reflection of viability (26).


Figure 1
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  		Fig. 1. Time course of intracellular ATP level during metabolic inhibition (MI) and washout of metabolic inhibitors in human cortical renal epithelial (HCRE) cells. On washout the solution containing the metabolic inhibitors was replaced by normal saline solution (see MATERIALS AND METHODS). ATP levels are indicated as % from the control, represented by the mean of ATP concentration from 2 samples per point. Values are means ± SE from 4 different monolayers.

 
In HCRE cells MI induced a transient increase in [Ca2+]i ({circ}, Fig. 2). The initial increase in [Ca2+]i occurred within 3.45 min (from 39.6 to 136.5 ± 15.8 nM) and was followed by a recovery phase, in which [Ca2+]i partially recovered and stabilized at 62.7 ± 7.3 nM. During the washout period [Ca2+]i recovered to 39.4 ± 8.1 nM in 10 min. It is worthwhile to note that despite the considerable energetic deficit the [Ca2+]i transient occurred in the presence of nonnegligible amounts of ATP. A second exposure to MI did not induce a transient increase in [Ca2+]i (peak type) but a continuous increase up to a level of 74.9 ± 11.9 nM.


Figure 2
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  		Fig. 2. Effect of MI on the cytosolic Ca2+ concentration ([Ca2+]i) in HCRE cells in normal Ca2+ environment ({circ}), n = 8 and in a Ca2+-free medium (bullet), n = 4. On washout the solution containing the metabolic inhibitors was replaced by normal saline solution (see MATERIALS AND METHODS). In the Ca2+-free medium experiment, the cells were preincubated for 5 min in the Ca2+-free medium before exposure to MI (not shown). [Ca2+]i values are indicated as means ± SE. SE values are depicted in dotted lines.

 
To reveal the factors responsible for the generation of the transient increase in [Ca2+]i, first we established the source of Ca2+ (extracellular vs. intracellular). When HCRE cells were subjected to MI in a Ca2+-free medium, the MI-induced increase in [Ca2+]i was reduced, accounting only for ~17 nM, from 4.4 ± 1.4 to 21.2 ± 2.8 nM (bullet, Fig. 2) and suggesting that the increase in [Ca2+]i was mainly originated from outside the cells. Secondly, to rule out the possibility that this transient elevation in [Ca2+]i could have been induced only by antimycin A or 2-deoxyglucose alone, [Ca2+]i was monitored in the presence of these inhibitors separately. As shown in Fig. 3, neither in the case of antimycin A ({triangleup}) nor in the case of 2-deoxyglucose alone ({blacktriangleup}) was a transient increase in [Ca2+]i noticed.


Figure 3
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  		Fig. 3. Effect of individual metabolic inhibitors on the evolution of [Ca2+]i: 10 µM antimycin A ({triangleup}), n = 4 and 10 mM 2-deoxyglucose ({blacktriangleup}), n = 4. Arrow indicates the moment when the metabolic inhibitors were added. [Ca2+]i values are indicated as means ± SE. SE values are depicted in dotted lines.

 
Which Ca2+ entry pathways could be involved in the noticed transient [Ca2+]i elevation? Taking into account that the major route for Ca2+ entry in nonexcitable cells is represented by the store-operated Ca2+ channels (SOCs) (37) and a previous report suggesting that these channels mediate hypoxia induced Ca2+ entry in proximal tubule cells (41), MI was applied in the presence of La3+, a blocker of SOCs. Figure 4A shows that MI induced a significantly (P < 0.01) smaller initial transient increase in [Ca2+]i in the presence of La3+, of only ~30 nM compared with the control of ~100 nM ({circ}) (Fig. 2). Importantly, the transient increase in [Ca2+]i was still distinct. These results suggest that SOCs are among the Ca2+ entry pathways in HCRE cells subjected to MI. Furthermore, we also observed that diltiazem, a blocker of L-type voltage-operated Ca2+ channels (VOCs), did not modify the transient [Ca2+]i rise (n = 3; data not shown).


Figure 4
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  		Fig. 4. [Ca2+]i changes during MI in HCRE cells. A: in the presence of 0.2 mM LaCl3. The peak value of [Ca2+]i is significantly decreased compared with the control, P < 0.01, Mann -Whitney U-test, n = 4. B: in the presence of 1 µM carbonyl-cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Before the exposure to MI, the cells were preincubated with FCCP for 10 min, n = 4.

 
Interestingly, the transient increase in [Ca2+]i was blocked by FCCP, a protonophore that dissipates the proton gradient across the inner mitochondrial membrane. When cells were preincubated with FCCP for 5 min and then subjected to MI in the continuous presence of FCCP, the transient increase did not occur and [Ca2+]i steadily increased during MI, reaching 98.6 ± 18.1 nM at the end of the MI period (Fig. 4B). Notice that when FCCP is applied, during the preincubation period, a peak-type response in [Ca2+]i occurs, which is likely correlated with the inactivation of the mitochondrial Ca2+ uniporter.

Furthermore, we focused on the recovery phase of the [Ca2+]i transient increase. To indirectly estimate the role of plasma membrane Na+/Ca2+ exchanger in the Ca2+ clearance, [Na+]i was monitored during MI. As expected, MI elicited an increase in [Na+]i, from 20 ± 2.3 mM to a plateau value of 61.2 ± 12.4 mM (3.5 times) ({circ}, Fig. 5). The decrease of the transmembrane Na+ gradient does not favor the activity of the Na+/Ca2+ exchanger. Another factor that could account for the observed [Ca2+]i clearance is the ER Ca2+ pump. To investigate the implication of ER Ca2+ pump, HCRE cells were preincubated for 15 min in the presence of thapsigargin, a blocker of the ER Ca2+ pumps and afterward subjected to MI in the continuous presence of this compound (Fig. 6A). Thapsigargin released ER Ca2+ (data not shown), and after a 15-min preincubation the [Ca2+]i stabilized to 104.4 ± 18.8 nM. When applied, MI induced a slight increase in [Ca2+]i, to 120.7 ± 19.7 nM, which was reached in 2.5 min. This increase was followed by a decrease in [Ca2+]i to a stable level of ~72 ± 11 nM, value similar with the level of [Ca2+]i at the end of recovery period in the absence of thapsigargin ({circ}, Fig. 2).


Figure 5
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  		Fig. 5. Changes in cytosolic Na+ concentration ([Na+]i) during MI in a normal Na+ environment ({circ}), n = 5; in the absence of external Na+ ({square}), n = 4; and in the absence of Na+ and the presence of CGP-37157 ({triangleup}), n = 6. For the experiments performed in the absence of Na+, the cells were incubated in a Na+-free medium (without or with CGP-37157) for 10 min before and during the treatment with metabolic inhibitors (not shown). [Na+]i values are indicated as means ± SE. SE values are depicted in dotted lines.

 

Figure 6
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  		Fig. 6. [Ca2+]i changes during MI in HCRE cells. A: in the presence of thapsigargin, the cells were preincubated for 15 min with 1 µM thapsigargin before exposure to MI (not shown), n = 7. B: in the absence of external Na+, the cells were incubated in a Na+-free medium for 10 min before and during treatment with metabolic inhibitors, n = 4. [Ca2+]i values are indicated as means ± SE. SE values are depicted in dotted lines.

 
On the basis of the similarities of the transient increase in [Ca2+]i seen in MDCK cells (2, 46) and in HCRE cells subjected to MI (present data), we hypothesized that mitochondrial NCX is involved in the [Ca2+]i recovery noticed in HCRE cells. To test this hypothesis, we first sought to find whether the [Ca2+]i recovery during MI is Na+ dependent. In a Na+-free medium, MI induced a continuous increase in [Ca2+]i and the recovery pattern was not present (Fig. 6B). Secondly, we directly investigated the role of mitochondrial NCX in the [Ca2+]i recovery, by using CGP-37157, a selective blocker of mitochondrial NCX. When MI was applied in the presence of CGP-37157, [Ca2+]i increased from 65.3 ± 3.9 nM, the value reached at the end of the 30 min preincubation period, to 159.4 ± 23.6 nM in 2.5 min (bullet, Fig. 7). This increase was followed by an incomplete recovery in [Ca2+]i, reaching 107.3 ± 6.3 nM at the end of the observation period. To facilitate the comparison, in Fig. 7 is also shown the control experiment: the time course of [Ca2+]i in the absence of CGP-37157 ({circ}).


Figure 7
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  		Fig. 7. Effect of MI on the [Ca2+]i in the presence of 25 µM CGP-37157 (bullet), n = 5. CGP-37157 was present 30 min before as well as during MI. For enabling the comparison, the control experiment ({circ}) trace was translated up with 11.36 nM Ca2+. After 10 min of MI, the differences between [Ca2+]i values are statistically significant (P < 0.01, Mann-Whitney U-test). [Ca2+]i values are indicated as means ± SE. SE values are depicted in dotted lines.

 
To investigate the ability of mitochondrial NCX to reverse during MI and to indirectly estimate the availability of mitochondrial Na+ to sustain the exchange, the MI experiment was repeated in a Na+-free medium (Fig. 5) and [Na+]i monitored. In these conditions, [Na+]i increased about fourfold in the first 5 min, from 10.4 ± 5 to 37.1 ± 9.9 mM ({square}). Notably this increase was completely blocked by CGP-37157 ({triangleup}), which indicates that the source of Na+ is mitochondria. Taken together, these data suggest that the mitochondrial NCX is of major significance in the Ca2+ clearance seen in HCRE cells.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our data show a biphasic behavior of [Ca2+]i during MI in HCRE cells (Fig. 2). This feature of the Ca2+ response has also been observed in MDCK cells (46) and in primary cultures of rat proximal tubular epithelial cells (7). In both cases, the [Ca2+]i recovery during MI was shown to have a protective effect against ischemic damage. Neither the mitochondrial respiration arrest with antimycin A nor the glycolysis blockade with 2-deoxyglucose induced a transient increase in [Ca2+]i (Fig. 3), ruling out a possible effect of either agent acting individually and suggest that only their combined effects induce such a transient increase in [Ca2+]i. The transient rise in [Ca2+]i is not seen in the presence of either antimycin A or 2-deoxyglucose alone, most likely because of a different cell ATP depletion level. Unlike antimycin A, 2-deoxyglucose reduces cellular ATP levels in two ways. It decreases ATP generation by acting as a competitive inhibitor of glycolysis, and it depletes existing ATP stores by undergoing extensive phosphorylation (26). Thus 2-deoxyglucose reduces cell ATP stores faster than antimycin A, a fact that is suggested also by the different [Ca2+]i evolution (Fig. 3). When both metabolic inhibitors are applied simultaneously, the ATP depletion is fast and more pronounced than in the presence of either alone. In these conditions the transient increase in Ca2+ is triggered.

MI-induced [Ca2+]i increase is mainly due to the Ca2+ influx.

In the present experiments the Ca2+ influx originated mainly from the external milieu as the omission of Ca2+ abolishes the MI-induced Ca2+ increase (Fig. 2). However, MI induces also Ca2+ release from the internal stores. Although VOCs were also reported to be involved in hypoxia-induced Ca2+ increase, the major route for Ca2+ entry in nonexcitable cells is the SOCs (37). In renal epithelial proximal cells both blockers of SOCs (1, 41) and VOCs (1, 41, 47) were shown to inhibit the Ca2+ increase induced by hypoxia. In renal epithelial distal cells neither verapamil nor nifedipine, VOCs blockers, could inhibit the Ca2+ increase induced by simulated ischemia (34). The present report suggests that the MI-induced Ca2+ entry in HCRE cells is mediated by SOCs. La3+, a SOC blocker (29), significantly decreased the Ca2+ entry (Fig. 4A). Moreover, no modification in the [Ca2+]i time course during MI occurred in HCRE cells in the presence of diltiazem, a blocker of VOCs; thus it is unlikely that these channels mediate the Ca2+ entry. Although the contribution of other calcium channels, such as the La3+-sensitive transient receptor potential channels, to Ca2+ entry in HCRE cells during MI cannot be excluded, SOCs remain as the main candidates. Indirectly, this conclusion is further supported by the fact that after the depletion of ER Ca2+, MI failed to induce an increase in [Ca2+]i (Fig. 6A). Classically, Ca2+ entry via SOCs is triggered by the ER Ca2+ release (38). After the thapsigargin preincubation period, the ER Ca2+ stores are already depleted, and the [Ca2+]i level is the consequence of a new equilibrium between Ca2+ entry and Ca2+ extrusion out of the cells. Moreover, it was reported that the activity of ER Ca2+ pumps controls the gating of SOCs (29). Another argument in favor of SOCs as mediators of Ca2+ entry in HCRE cells subjected to MI relies on the fact that mitochondria can take up Ca2+ entering this way (20, 22).

During the second exposure to MI, the transient increase in [Ca2+]i did not occur (Fig. 2). Most likely this happens as a consequence of the impairment of mitochondrial signaling capacity and/or the ATP depletion status (~17% from the control) (Fig. 1). The connection between SOCs-mediated Ca2+ entry and ATP depletion was established in lymphocytes (33), in HeLa, Jurkat, and Erlich ascites tumor cells as well in hepatocytes (14). In these two studies, ATP depletion was proven to inhibit the capacitative Ca2+ entry. Moreover, in line with these findings, ADP was shown to inhibit Ca2+ release-activated Ca2+ current in a basophilic cell line (24).

The first phase of the transient MI-induced [Ca2+]i increase depends on energized mitochondria.

When mitochondria were deenergized, the time course of the [Ca2+]i increase during MI was different (Fig. 4B). In these conditions [Ca2+]i rises steadily, suggesting that energized mitochondria are responsible for the first phase of the transient [Ca2+]i increase. It is widely accepted that [Ca2+]i modification during MI is bound to the level of ATP loss, a link established mainly by the ATP-fueled Ca2+ pumps (4, 28, 39). On the other hand, there is evidence to show that the ATP level is not the only factor involved in Ca2+ regulation during MI (50). Whereas in MDCK cells the transient increase in [Ca2+]i occurs in the absence of ATP (46), in HCRE cells the ATP level is significant during this process (Fig. 1). This suggests, at least in HCRE cells, that ATP level does not directly determine the first phase of the transient [Ca2+]i increase. In agreement with this observation, in Jurkat cells, Ca2+ entry via SOCs was shown to be depressed owing to mitochondria uncoupling and not related to the cellular ATP-to-ADP ratio (30). These findings indicate that short-time MI effects may occur through more complicated signaling pathways rather than through the direct inhibition of ATP production. Although similar findings were reported in T lymphocytes (21, 22) as well as in glioma C6, Ehrlich ascites tumor cells, and human fibroblasts (52), where mitochondrial uncouplers inhibited the rate of Ca2+ entry via SOCs, this is the first report showing an association between MI and a possible mitochondrial signaling effect.

Possible MI-induced signaling events mediated by mitochondria.

Recent evidence points to a dynamic interplay between mitochondria, ER, and the plasma membrane in store-operated Ca2+ entry (15, 22, 32, 36, 37). In this work, the data show that in HCRE cells, energized mitochondria are required to mediate the Ca2+ entry during the first phase of MI, probably via SOCs. Hence it seems that there is a cross talk between mitochondria and the structures responsible for Ca2+ entry. Interestingly, this communication is impaired during the second exposure to MI, when [Ca2+]i increase is continuous (Fig. 2) and shows similarities with [Ca2+]i increase in the presence of FCCP (Fig. 4B). In agreement with this observation, it was shown that ATP depletion inhibits the SOC-mediated Ca2+ entry (14, 24, 33). Finally, it is well documented that the mitochondrial activity is severely impaired in proximal tubules subjected to hypoxia-reoxygenation (53, 54), which could be the overall source of the lack of communication.

In endothelial cells it was recently suggested that Ca2+ entering via SOCs transits mitochondria before being delivered to ER and that this has an impact on Ca2+ signaling and Ca2+ homeostasis (31, 37). The present data in this work does not address the role of ER in this particular signaling process directly. However, in the presence of thapsigargin, MI induced only a slight increase in [Ca2+]i, only ~13 mM (Fig. 6A). Nevertheless, the starting level was higher (~2.5 times) than the basal [Ca2+]i. It is possible that the lack of MI-induced [Ca2+]i increase in the presence of thapsigargin is due either to the impairment of capacitative calcium entry itself (29) or to the suppression of communication between ER and mitochondria. It is worth noting that FCCP depolarizes the mitochondrial membrane and thus releases mitochondrial Ca2+. This process is shown in the Fig. 4B, where FCCP alone induces a short transient increase in [Ca2+]i. Whether mitochondrial Ca2+ depletion is the explanation for the lack of communication still has to be investigated.

What can be the role of energized mitochondria dependent MI in the induced [Ca2+]i increase? Besides general inhibition of protein synthesis, hypoxia upregulates the genes responsible for the adaptation to this stress (19). This process is mediated by the hypoxia-inducible factors (HIFs). In renal epithelia, HIF-1 is considered the key mediator of acute hypoxic signaling (42, 43). The link between the activation of these factors and the onset of the hypoxic event could be associated with the [Ca2+]i elevation. In line with this suggestion, a recent report shows that the transcription activation of HIF proteins is dependent on the hypoxia-induced Ca2+ influx (23). On the other hand, mitochondria-dependent [Ca2+]i elevation could be seen as an event involved in the process of oxygen sensing, in which mitochondria have a dominant role (51).

Mitochondrial NCX is involved in the Ca2+ clearance during MI.

The recovery phase of the transient [Ca2+]i increase can be due to the Ca2+ extrusion out of the cells, via ATP-fueled plasma Ca2+ pumps or plasma membrane NCX and/or buffering capacity of internal stores, such as ER and/or mitochondria. Besides being a blocker of SOCs, La3+ is a potent blocker of plasma membrane Ca2+ pumps (5, 6). The recovery of the La3+ sensitive [Ca2+]i increase is not influenced by La3+, and thus the Ca2+ clearance in this particular condition is unlikely to be mediated by plasma Ca2+ pumps (Fig. 4A). However, the data cannot provide information about the clearance of the La3+ insensitive [Ca2+]i increase, which could be mediated by these pumps. The activity of plasma membrane Na+/Ca2+ exchanger is expected to be diminished during MI, as the consequence of the increase in [Na+]i (Fig. 5). Finally, ER appears to be not responsible for the calcium clearance seen during MI in HCRE cells (Fig. 6A). As discussed, the ER depletion in Ca2+ disables probably the capacitative Ca2+ entry at the onset of MI. In these conditions, although Ca2+ overload is generated mainly by the ER incapacity to take up Ca2+, the [Ca2+]i recovery process is still present.

Mitochondria can take up Ca2+ by means of the mitochondrial Ca2+ uniporter and via the mitochondrial NCX. The activity of the Ca2+ uniporter relies on the high {Delta}{Psi} maintained across the inner mitochondrial membrane by mitochondrial respiration. When {Delta}{Psi} collapses, mitochondrial Ca2+ is released. This is shown in Fig. 4B, where a peak-type response in [Ca2+]i follows the addition of FCCP. During MI, because of the gradual collapse of {Delta}{Psi}, the uniporter contribution in the Ca2+ clearance is reduced and less and less important whereas the energetic deficit advances (2, 46, 53). The blockage of mitochondrial NCX does not significantly modify the increase in [Ca2+]i during MI, but the recovery efficiency was different (Fig. 7). Moreover, like MDCK cells, HCRE cells show a Na+-dependent Ca2+ clearance (Fig. 6B). The similarities between [Ca2+]i increase in the presence and in the absence of CGP-37157 point out that mitochondria are not the source of Ca2+, because the normal route of Ca2+ exit from mitochondria, the mitochondrial NCX, is blocked. In turn, it is likely that mitochondria take up Ca2+ during the recovery phase via mitochondrial NCX working in the entry mode, as showed in MDCK cells (2, 46). According to these reports, the activity of mitochondrial NCX during the second phase of MI-induced transient Ca2+ increase, and thus the Ca2+ buffering capacity, depends on suitable amounts of cytosolic and mitochondrial Na+. By the use of CoronaRed, a probe for mitochondrial Na+, it was showed that in a normal Na+ environment, MI induces a sustained increase in mitochondrial [Na+] which peaks about at the onset of [Ca2+]i recovery (2). This increase was followed by a slow decrease, corresponding to the [Ca2+]i recovery period and sustained by the activity of mitochondrial NCX working in reverse mode. In the present study, after 10 min of preincubation in a Na+-free medium and when [Na+]i is decreased to about half of the control, MI induces in HCRE cells a fourfold increase in [Na+]i in the first 5 min (Fig. 5). Notably, this increase is completely abolished when the mitochondrial NCX is blocked. Thus, in a Na+-free medium, MI triggers the activity of the mitochondrial NCX, which exchanges cytosolic Ca2+ for the available mitochondrial Na+. It is instructive to examine the differences between the two situations. First, whereas in a normal Na+ environment the MI-associated rise in [Na+]i induces an increase in mitochondrial Na+ that will fuel the recovery in [Ca2+]i, in a Na+-free medium the recovery fails as the consequence of low stores of mitochondrial Na+. In a Na+-free medium, it is possible to consider the rate of [Na+]i increase and respective decrease connected with the rate of mitochondrial NCX exchange. In this light, during MI, the steady decrease in [Na+]i after the peak is the result of a continuous decrease in mitochondrial Na+ concentration (Fig. 5). Secondly, in a Na+-free medium, the mitochondrial NCX reverses from the beginning, as the consequence of a favorable Na+ gradient between the mitochondrial matrix and cytosol. Although it is difficult to draw definitive conclusions about the intimate exchange process, these experiments clearly show that the mitochondrial NCX can reverse during MI.

Taking into account that [Ca2+]i recovery during MI was impaired in HCRE cells in the presence of CGP-37157 and abolished in the absence of external Na+, on the basis of the similarities reported in MDCK cells (2, 46) mitochondrial NCX appears to be one of the factors involved in Ca2+ clearance. This reversal of mitochondrial NCX during hypoxia has also been observed in rat cardiomyocytes (16, 17).

Summary.

Similarly to the previous findings in MI challenged MDCK cells (46), in HCRE cells [Ca2+]i shows a transient increase during MI. The first phase of the [Ca2+]i transient increase is dependent on the preservation of the inner mitochondrial potential, it is likely mediated by SOCs, and it seems to represent a mitochondria-mediated signaling event, triggered by the onset of MI. The [Ca2+]i recovery phase partially depends on the activity of mitochondrial NCX, which can reverse during MI. These results show that in human renal epithelial proximal cells subjected to MI mitochondria play an important role in calcium regulation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
A. Caplanusi was supported by a Fulbright Senior Fellowship Award. Research was supported by a National Heart, Lung, and Blood Institute Grant (HL-18426), a COBRE grant (P20 RR-017659) from the Institutional Developmental Award (IdeA) program of the National Center for Research Resources, and a Millennium Health Excellence Fund from the Louisiana Board of Regents [HEF (2001-06)-07]. The Southeast Louisiana Veterans Healthcare System provided salary (T. G. Hammond) in support of these studies.


   ACKNOWLEDGMENTS
 
We thank Drs. Sanda Clejan, Stefan Constantinescu, Philippe Gailly, Nazih Nakhoul, Patrick Van Der Smissen, and Paul Steels for the stimulating discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Caplanusi, Cell Biology Dept., Université Catholique de Louvain (UCL) and Christian de Duve Institute of Cellular Pathology (ICP), UCL-7541, 75 av. Hippocrate, B-1200 Bruxelles, Belgium

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


   REFERENCES
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 ABSTRACT
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 DISCUSSION
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