Bone Marrow Mononuclear Cells Restore Normal Mitochondrial Ca 2+ Handling and Ca 2+ -Induced Depolarization of the Internal Mitochondrial Membrane by Inhibiting the Permeability Transition Pore After Ischemia/Reperfusion

Abstract

Acute kidney injury due to ischemia followed by reperfusion (IR) is a severe clinical condition with high death rates. IR affects the proximal tubule segments due to their predominantly oxidative metabolism and profoundly altered mitochondrial functions. We previously described the impact of IR on oxygen consumption, the generation of membrane potential (ΔΨ), and formation of reactive oxygen species, together with inflammatory and structural alterations. We also demonstrated the benefits of bone marrow mononuclear cells (BMMC) administration in these alterations. The objective of the present study has been to investigate the effect of IR and the influence of BMMC on the mechanisms of Ca²⁺ handling in mitochondria of the proximal tubule cells. IR inhibited the rapid accumulation of Ca²⁺ (Ca²⁺ green fluorescence assays) and induced the opening of the cyclosporine A-sensitive permeability transition pore (PTP), alterations prevented by BMMC. IR accelerated Ca²⁺-induced decrease of ΔΨ (Safranin O fluorescence assays), as evidenced by decreased requirement for Ca²⁺ load and t_1/2 for complete depolarization. Addition of BMMC and ADP recovered the normal depolarization profile, suggesting that stabilization of the adenine nucleotide translocase (ANT) in a conformation that inhibits PTP opening offers a partial defense mechanism against IR injury. Moreover, as ANT forms a complex with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, it is possible that this complex is also a target for IR injury—thus favoring Ca²⁺ release, as well as the supramolecular structure that BMMC protects. These beneficial effects are accompanied by a stimulus of the citric acid cycle—which feed the mitochondrial complexes with the electrons removed from different substrates—as the result of accentuated stimulus of citrate synthase activity by BMMC.

Keywords

renal ischemia/reperfusion bone marrow mononuclear cells permeability transition pore mitochondrial membrane potential

Introduction

Renal ischemia followed by reperfusion (IR) is characterized by an initial restriction of renal blood flow, followed by restoration of circulation and O₂ supply^1,2. The sequence of IR events is very common in acute kidney injury (AKI), one of the most severe clinical conditions in intensive care units worldwide³. Death from AKI is very frequent; more than 1.7 million of deaths per year are caused by AKI, with 1.4 million in low- and middle-income countries⁴. Survivors frequently develop chronic kidney disease (CKD)⁵, a risk factor for AKI⁶.

Renal proximal tubule cells are particularly vulnerable to IR because of their intense oxidative metabolism, mitochondria from these cells being particularly sensitive to IR injury⁷. In the absence of O₂, depletion of ATP rapidly occurs, leading to cell death with a concomitant and a sudden decrease in the glomerular filtration rate and deregulation of the renal hemodynamics as a whole⁸. During reoxygenation, an important burst of reactive oxygen species (ROS) occurs as a result of an uncontrolled premature transference of electrons to O₂ to form $O_{2}^{• -}$ in steps before physiological transfer at the level of cytochrome oxidase^9,10. A high production of ROS is also the result of Ca²⁺ release from the intra-mitochondrial compartment, followed by activation of mitochondrial Ca²⁺-dependent cytosolic proteases, the xanthine oxidase pathway, and NADPH oxidases, though the latter are considered secondary to the initial mitochondrial burst of mitochondrial $O_{2}^{• -}$ ¹¹.

The central role of uncontrolled Ca²⁺ handling in the opening of the permeability transition pore (PTP) during IR, in the collapse of mitochondrial membrane potential (ΔΨ) and in oxidant damage, is widely accepted^12,13. However, the molecular mechanisms by which IR alters mitochondrial Ca²⁺ homeostasis and the Ca²⁺-induced alterations of ΔΨ, in an environment of exacerbated production of $O_{2}^{° -}$ and other ROS, are not fully understood. Answers to questions are far from clear: What are the mechanisms by which cell therapies—a promise that emerged over the two last decades in the prevention and regeneration of renal lesions^14–18—could be beneficial in preventing or repairing mitochondrial damage in IR? Are these potential mechanisms associated with preservation of mitochondrial Ca²⁺ transport mechanisms and physiological Ca²⁺-induced modulation of ΔΨ?

We previously described the influence of renal IR on electron fluxes, generation of the ΔΨ, ATP synthesis, ROS generation, S-nitrosylation of proteins in renal cells, apoptosis, and inflammatory responses. We also investigated the effects of bone marrow mononuclear cells (BMMC) in these processes, correlated to the recovery of tubular lesions¹⁹. The beneficial properties of BMMC, which have been centrally used in renal regenerative medicine for a number of years^15,20–22, remerged as promising again since the discovery of the potential therapeutic role of extracellular vesicles (EVs) secreted^23,24 by the most abundant parcel of their cell population, CD11⁺ CD29^– granulocytes from the CD45⁺ cluster¹⁹.

The object of the present study has been to investigate how renal IR modifies Ca²⁺ transport mechanisms in renal mitochondria in rats, together with the Ca²⁺-induced depolarization of the internal mitochondrial membrane, and to see whether BMMC can recover—or prevent—the possible functional alterations provoked by acute IR. Participation of the Ca²⁺-provoked opening of PTP—with the possible beneficial influence of BMMC—has been investigated. As the electrons removed from the substrates oxidized in the citric acid cycle feed the mitochondrial transport system, we also investigated the impact of IR and the effects of BMMC on the citrate synthase activity. We chose this enzyme because it catalyzes the supply of 2C fragments from acetyl-CoA (AcCoA) at the first step of the cycle²⁵.

Materials and Methods

Animals

Adult male Wistar rats (180–200 g) obtained from the vivarium of the Leopoldo de Meis Institute of Biochemistry at Federal University of Rio de Janeiro and Oswaldo Cruz Institute (Rio de Janeiro) were housed at 22°C in ventilated cages under humidity control and 12 h:12 h light/dark cycle, receiving commercial chow (Purina Agribands, Paulínia, Brazil) and filtered water ad libitum. The experimental protocols followed the guidelines of the local Committee of Ethics in the Use of Animals for Research (approval number: 104).

After 1 week of acclimatization, the animals were divided into three groups: (i) SHAM with opening of the abdominal cavity and smooth manipulation of the renal pedicles, (ii) IR: bilateral clamping of the renal arteries with silver clamps for 30 min followed by 1 h of reperfusion (one specific experiment was carried out after 24 h of reperfusion), and (iii) IR rats that received subcapsularly 1 × 10⁶ bone marrow–derived mononuclear cells (IR + BMMC) 1 h before arterial clamping. Before surgery the animals were anesthetized by intraperitoneal injection of 0.4 ml of a solution containing 5% w/v ketamine hydrochloride (Cristália, Itapira, Brazil) and 0.02 mg/ml xylazine hydrochloride (Bayer SA, São Paulo, Brazil) (proportion 3:1). The rats in the groups given 24 h reperfusion were sutured with cotton thread 3.0, locally treated with lidocaine to suppress pain, and returned to individual cages. All animals were killed by decapitation.

Cells

The source of cells was bone marrow, which was obtained by flushing the femurs and tibias with saline of rats of the same gender and age, and with similar body mass to those used to compose the three experimental groups given above. Isolation of BMMC was carried out by using Histopaque® (Sigma-Aldrich, Saint Louis, MO, USA). Bone marrow samples were initially centrifuged for 10 min at 395 × g (25°C) and the recovered sediment resuspended in 3.5 ml DMEM without serum, homogenized, layered on to 3.5 ml Histopaque®, and centrifuged at 403 × g for 30 min (initial acceleration and final deceleration lasting 120 and 150 s to preserve the gradient). The cells localized in the interface between DMEM and Histopaque® were carefully removed, suspended in DMEM, recentrifuged at 395 × g, suspended again in DMEM, and used within 2 h. Viability was assessed with trypan blue. The immunophenotype of the cells was characterized as described in the previous studies^19,23,24,26. Their viability was also assessed with trypan blue. Four subpopulations were characterized from the cluster of differentiation 45 (CD45⁺) cell population (95% of total cells): (i) T helper lymphocytes (0.15%), (ii) T cytotoxic lymphocytes (1.5%), (iii) monocytes (13%), and (iv) granulocytes (54%)¹⁹. For a fuller illustration of the phenotype characterization of the used BMMC, see, for example, Fig. 1 in Beiral et al.¹⁹ Controls for tracing of BMMC in the cortical renal parenchyma were carried out with amine-reactive Cell Trace™ Far Red DDAO-succinimidyl ester (Invitrogen, Grand Island, NY, USA) to obtain three-dimensional (3D) reconstructions visualized in an ApoTome microscope (ApoTome Axio Imager M2, Carl Zeiss, Inc., Jena, Germany)¹⁹.

Figure 1.

Rapid mitochondrial Ca²⁺ uptake is inhibited by IR and totally recovered by BMMC treatment. Representative recordings of Ca²⁺ green fluorescence (extra-mitochondrial Ca²⁺) after a single pulse of 80 µM CaCl₂ (160 nmol Ca²⁺ in a 2 ml cuvette) to mitochondria suspended in MIR05 (0.5 mg/ml) and energized with 10 mM succinate in the presence of 100 µM ADP. Mitochondria from SHAM (A), IR (B), and IR + BMMC (C) were isolated and assayed after 1 h of reperfusion. In the case of the SHAM rats, which had only gentle manipulation of the vascular renal pedicle, the time of “reperfusion” refers to a time that was the same as for the IR and IR + BMMC animals after release from clamping. IR: ischemia followed by reperfusion; BMMC: bone marrow mononuclear cells.

Mitochondrial Isolation

Each pair of kidneys was rinsed with a cold solution containing 250 mM sucrose, 10 mM HEPES–Tris (final pH 7.4), 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.15 mg/ml trypsin inhibitor (Sigma-Aldrich), which was used in all the following steps. The cortex corticis, part of renal tissue where >90% of the cell population corresponds to proximal tubules²⁷, was rinsed and then homogenized by hand through 11 cycles in a 30 ml glass homogenizer (Wheaton Sci., Wheaton, IL, USA). After centrifugation at 600 × g for 5 min to remove unbroken cells, nuclei, and cell debris, the supernatant was centrifuged at 12,000 × g and the sediment washed twice in 10 ml of the above solution at the same speed, finally being suspended in 0.3 ml of the same solution containing 10 mg/ml bovine serum albumin (BSA; fatty acid-free; Sigma-Aldrich) and used immediately. Protein content was quantified by Folin phenol reagent²⁸.

Mitochondrial Ca²⁺ Accumulation and Release

Calcium uptake was measured by using a Calcium green 5N (Life Technologies, Carlsbad, CA, USA) probe and a Hitachi fluorometer model F-3010 (Hitachi Ltd., Tokyo, Japan) at 506 nm (excitation)/532 nm (emission). The assay medium (37°C) contained 0.5 mg/ml of mitochondria, MIR05 without EGTA (110 mM sucrose, 3 mM MgCl₂, 20 mM taurine, 60 mM MES, 20 mM HEPES, 10 mM KH₂PO₄, and 1 mg/ml BSA), pH adjusted to 7.1 by addition of KOH, and supplied with 100 µM ADP, 0.2 µM Calcium green, and 1 µM rotenone. Recordings in the presence or absence of 1 µM cyclosporine A (CsA) were initiated just before addition of 10 mM succinate and successive pulses of 80 µM CaCl₂ at 2-min intervals. After a fast increase of fluorescence that corresponded to Ca²⁺ binding to the extra-mitochondrial Calcium green, a decrease with two components occurred (see “Results” section): a very fast one—which cannot be resolved by this method—and a slower one. The rate of this component was measured by determination of the slope over 20 s from the fluorescence peak. After several additions, the uptake stopped and a sudden release occurred.

Ca²⁺-Induced Depolarization of the Inner Mitochondrial Membrane

The Ca²⁺-induced modifications of mitochondrial transmembrane potential (ΔΨ) were measured by the evolution of fluorescence of Safranin O (Sigma-Aldrich) at 495 nm (excitation)/586 nm (emission) in the presence of energized mitochondria. The mitochondria were preincubated for 2 min in the chamber of the Hitachi F-3010 fluorometer at 37°C with a solution containing 320 mM mannitol, 10 mM Tris HCl (pH 7.4), 8 mM Tris-phosphate (H₃PO₄ neutralized with Tris base up to pH 7.4), 4 mM MgCl₂, 1 mg/ml BSA fatty acid-free (Sigma-Aldrich), 1 µM rotenone (Sigma-Aldrich), and 5 µM Safranin O (Sigma-Aldrich), in the absence or presence of 1 mM ADP. After mitochondrial energization with 10 mM succinate and rapid decrease in fluorescence intensity, successive pulses of 10 µM CaCl₂ allowed measurements of total Ca²⁺ load ([Ca²⁺]_t) required for complete depolarization (completed by adding 1 µM FCCP) and of the time necessary for 50% recovery of the baseline (t_1/2).

Citrate Synthase Activity

Total citrate synthase activity was measured by following the formation of 5-thio-2-nitrobenzoic acid (TNB) during the irreversible colorimetric reaction:

C o A - S H + D T N B \to T N B + C o A - S - S - T N B,

coupled in vitro to the reaction catalyzed by mitochondrial citrate synthase:

O A A + A c C o A \to C i t r a t e,

where CoA-SH is coenzyme A, DTNB is 5,5′-dithiobis-(2-nitrobenzoic acid), OAA is oxaloacetate, and AcCoA is acetyl-CoA. The yellow product, TNB, was quantified by measuring absorbance (molar absorption coefficient 13.6 mM^–1 cm^–1) at 412 nm in a spectrometer UV-1800 UV-VIS (Shimatzu, Kyoto, Japan). The assays (at 25°C) were carried out in 1 ml of medium containing 0.1 mg/ml total mitochondrial protein preincubated for 2 min in 10 mM Tris HCl (final pH 8.5), 10 mM KH₂PO₄, 2 mM EDTA, 0.1% (w/v) Triton X-100, 0.1 mM AcCoA (Sigma-Aldrich), and 2 mM DTNB (Sigma-Aldrich). The reaction was started by adding 10 mM OAA (Sigma-Aldrich) and recorded over 10 min.

Statistical Analysis

Bar graphs and symbols represent means ± SEM (standard error of the mean). One-way analysis of variance (ANOVA) followed by Tukey’s test was used to assess differences among three mean values, and unpaired Student’s t test was used for the comparison of two means, as detailed in the corresponding figure legends or in the text. Correlation between two variables was assessed by the least squares method. P < 0.05 was considered statistically significant; P values are given within the panels of Figs. 5, 7, and 8, and in the legend to Fig. 6. Calculations were carried out using GraphPad Prism®, Version 6 (GraphPad Software, Inc., San Diego, CA, USA).

Results

IR Decreased Rapid Ca²⁺ Uptake by Mitochondria From Proximal Tubule Cells: Total Recovery by BMMC

Fig. 1 shows representative recordings of the time course of Ca²⁺ green fluorescence after a single addition of 80 µM Ca²⁺ to suspensions of mitochondria isolated 1 h after the beginning of reperfusion, or the equivalent time in SHAM rats. The traces show a very fast increase in Ca²⁺ green fluorescence followed by a slower decrease. This profile corresponds to the signal triggered by Ca²⁺ addition, followed by the entrance of the cation into the mitochondrial matrix. The height of the peak is lower in mitochondria from SHAM rats (Fig. 1A) than in mitochondria isolated from proximal tubules of rats submitted to IR (Fig. 1B), indicating that more Ca²⁺ remained transiently in the medium in this group. This means that a rapid mitochondrial Ca²⁺ uptake, which occurs in time intervals that cannot be resolved with this approach, became impaired as the result of the IR lesions. The time course of Ca²⁺ green fluorescence in IR rats that received subcapsular administration of BMMC 1 h before bilateral arterial clamping (IR + BMMC group) was similar to that in SHAM rats (Fig. 1C).

The differences in the evolution of Ca²⁺ green fluorescence are better perceived after successive Ca²⁺ pulses and quantification of the percent departure of fluorescence from the baseline 2 min after each addition. Increasing departure indicates that more Ca²⁺ remains in the medium after each uptake cycle. Fig. 2 shows representative recordings in the absence (A–C) and presence of 1 µM CsA (D–F). There is a progressive exponential increase of extra-mitochondrial Ca²⁺, which is more accentuated in the case of IR rats, and was recovered in the group receiving BMMC [compare circles in Fig. 2H with those in Fig. 2G (SHAM) and Fig. 2I (IR + BMMC)]. The values of percent departure from baseline were lower when CsA was present in the assay medium and more Ca²⁺ could be added before the sudden efflux, as demonstrated by the shift of the triangle symbols to 720 µM Ca²⁺ (compare with the corresponding circles at 560 µM Ca²⁺).

Figure 2.

Time course of mitochondrial Ca²⁺ accumulation after successive additions of Ca²⁺: effect of CsA. (A–C) Representative traces showing the evolution of Ca²⁺ green fluorescence after successive Ca²⁺ pulses in the absence of CsA. (D, E) Representative traces showing the evolution of Ca²⁺ green fluorescence after successive Ca²⁺ pulses in the presence of 1 µM CsA. Mitochondria (0.5 mg/ml) from SHAM (A, D), IR (B, E), or IR + BMMC (C, F) were incubated in Ca²⁺ green-containing media (MIR05) plus 100 µM ADP and energized with 10 mM succinate. The samples were successively supplied with CaCl₂ pulses (80 µM) at 2-min intervals. The final ascending part of the traces represents the sudden Ca²⁺ efflux from the intra-mitochondrial compartment. (G–I) Percent change of Ca²⁺ green fluorescence (departure from baseline) 2 min after each addition of CaCl₂ to give the Ca²⁺ concentrations shown on the abscissae, in the absence (circles) or presence (triangles) of 1 µM CsA. In some cases, the error bar is smaller than the symbol size. The smooth lines were adjusted to the experimental points (means ± SEM) by using the function y = y_oe^kx, where y corresponds to the percent increase of fluorescence from baseline per minute after each Ca²⁺ addition, y_o corresponds to the theoretical departure of fluorescence before the first Ca²⁺ addition, k is the constant of fluorescence increase (ie, the constant of extra-mitochondrial Ca²⁺ increase after each Ca²⁺ pulse), x is the cumulative Ca²⁺ concentration, and e has the usual meaning. Using one-way ANOVA followed by Tukey’s test, we compared the means (n = 5−7 different mitochondrial preparations) of fluorescence changes at 560 µM Ca²⁺ in the absence of CsA. The P values are given in the text. In the presence of CsA, the means of fluorescence changes (n = 5−8) were compared at 720 µM Ca²⁺, the P values also being given in the text. CsA: cyclosporine A; IR: ischemia followed by reperfusion; BMMC: bone marrow mononuclear cells; SEM: standard error of the mean; ANOVA: analysis of variance.

The rate constant, k, obtained with the use of the exponential function, y = y_oe^kx, allows quantitative comparison of the velocity by which ΔF (ie, extra-mitochondrial Ca²⁺) increases per minute after each Ca²⁺ addition. In the absence of CsA (circles), their values were 0.0032, 0.0051, and 0.0043 µM^–1 min^–1 for SHAM, IR, and IR + BMMC rats, respectively, indicating that 50% less Ca²⁺ were transported inside the renal mitochondria from IR rats and that a significant return toward SHAM rates occurred in the group receiving BMMC. Differences among the three experimental groups also emerged from a comparison of the ΔF at 560 µM Ca²⁺: P < 0.0001 (SHAM vs IR), P = 0.0244 (SHAM vs IR + BMMC), and P = 0.0009 (IR vs IR + BMMC). The changes provoked by CsA (triangles) are twofold. First, the rate constant k decreased in all groups; their values dropped to 0.0019 (SHAM), 0.0039 (IR), and 0.0024 µM^–1 min^–1 (IR + BMMC). Second, the difference in ΔF per minute at 720 µM Ca²⁺ between SHAM and IR + BMMC disappeared (P > 0.6437), that is, CsA contributed to the entire recovery of Ca²⁺ influx induced by BMMC in the absence of the drug. The values for the other comparisons were P = 0.0001 (SHAM vs IR) and P = 0.0003 (IR vs IR + BMMC).

The first-order and CsA-sensitive decrease in rapid Ca²⁺ entry into the renal intra-mitochondrial compartment in IR rats, as demonstrated by the greater height of the green fluorescence peak and the greater k value, was accompanied by modifications in two processes: one was related to the slower phase of Ca²⁺ uptake, and the other was related to the capacity of Ca²⁺ retention. Fig. 3A shows that the decrease in Ca²⁺ green fluorescence, which corresponds to the slow Ca²⁺ entry, was faster in mitochondria from IR rats (circles) than SHAM (squares) or IR + BMMC rats (triangles). This observation is additional evidence that less Ca²⁺ entered the mitochondria through the faster component and that intra-mitochondrial concentration was initially lower at the beginning of the slower uptake phase. Moreover, the tendency of Ca²⁺ efflux in IR mitochondria gradually rose from the beginning of the additions of Ca²⁺ (smooth curve in Fig. 3A). In the assays carried out in the presence of 1 µM CsA (Fig. 3B), the rates of slow Ca²⁺ entry were similar in the three groups and additional Ca²⁺ loads—which totaled 320 µM (55% more) in IR mitochondria—could be added before any tendency of efflux was established (see the shaded area in Fig. 3B).

Figure 3.

Rate of Ca²⁺ green fluorescence decay (ΔF/min) from the peaks obtained after each pulse of 80 µM CaCl₂ at 2-min intervals in media containing mitochondria (0.5 mg/ml), energized with 10 mM succinate and supplied with 100 µM ADP in the absence (A) or in the presence of 1 µM CsA (B). The rates were measured during the linear phase of decay (20 s) after each CaCl₂ pulse. The abscissae show the accumulated Ca²⁺ in the medium. The symbols correspond to SHAM (squares), IR (circles), and IR + BMMC rats (triangles). The dotted horizontal line at ΔF/min = 0 indicates Ca²⁺ influx = Ca²⁺ efflux, and negative values indicate that Ca²⁺ influx > Ca²⁺ efflux. Values are means ± SEM. In some cases, the error bars are smaller than the symbol size. The smooth continuous curve in (A) (circles), drawn by hand, reveals an initially very slow and then a sudden tendency to Ca²⁺ efflux. The shaded area in (B) indicates the extra [Ca²⁺] that can be provided to the medium containing IR mitochondria, when CsA is present, before the pulse that triggers the rapid Ca²⁺ efflux. In (A), points represent means of 4 (SHAM), 5 (IR), and 4 (IR + BMMC) determinations carried out by using different mitochondrial preparations. In (B), points represent means of 4 (SHAM), 6 (IR), and 4 (IR + BMMC) determinations, also carried out with the use of different mitochondrial preparations. In (A), asterisks indicate statistically different IR values with respect to SHAM and IR + BMMC (one-way ANOVA followed by Tukey’s test comparing Ca²⁺ concentration-matched values). No differences were found between SHAM and IR + BMMC across the range of Ca²⁺ concentrations. In (B), no differences were found among the three groups across the range of Ca²⁺ concentrations. CsA: cyclosporine A; IR: ischemia followed by reperfusion; BMMC: bone marrow mononuclear cells; SEM: standard error of the mean; ANOVA: analysis of variance.

IR Alters the Response to Ca²⁺ of Mitochondrial Membrane Potential: Effect of BMMC and ADP

IR-induced alteration in mitochondrial Ca²⁺ handling also had an impact on the Ca²⁺ response of the membrane potential of energized mitochondria (Figs. 4 and 5). The representative recordings of Safranin O fluorescence after Ca²⁺ addition were investigated in the absence (Fig. 4A–C) and in the presence of 1 mM ADP (Fig. 4D–F). After mitochondrial energization by addition of succinate (arrows), successive pulses of 10 µM Ca²⁺ provoked a progressive depolarization of the inner mitochondrial membrane, which was faster in mitochondria isolated from kidneys submitted to with IR than in the SHAM and IR + BMMC groups (compare Fig. 4B with Fig. 4A, C). As in the case of Ca²⁺ handling measurements (Figs. 2 and 3), administration of BMMC before the IR episode partially restored the depolarization profile to that of the SHAM group. Ca²⁺-induced depolarization was slower in the presence of ADP (Fig. 4D–F); however, it remained accelerated in IR mitochondria (compare Fig. 4E with Fig. 4D, F).

Figure 4.

Accelerated Ca²⁺-induced depolarization of inner mitochondrial membrane after IR. Representative recordings of Safranin O fluorescence after energization of mitochondria by addition of 10 mM succinate (arrows) in the absence (A–C) or presence of 1 mM ADP (D–F). Mitochondria (0.1 mg/ml) from SHAM (A, D), IR (B, E), or IR + BMMC (C, F) were incubated in Safranin O-containing medium (see “Materials and Methods” section) supplied with successive additions of CaCl₂ pulses (10 µM) at the times indicated on the abscissae (after the first arrow). In the experiments performed in the presence of ADP, it was supplied before the first addition of Ca²⁺. IR: ischemia followed by reperfusion; BMMC: bone marrow mononuclear cells.

Figure 5.

Ca²⁺-induced depolarization of the mitochondrial internal membrane. Ca²⁺ load required for total depolarization ([Ca²⁺]_t) (A, C) and time required for half-depolarization (t_1/2) (B, D). Assay conditions were as described in the legend to Fig. 4 and in the “Materials and Methods” section, in the absence (A, B) or presence of 1 mM ADP (C, D). Bars represent means ± SEM. Groups are indicated on the abscissae. Using one-way ANOVA followed by Tukey’s test assessed differences, which are indicated within the panels. Without ADP: SHAM, n = 6; IR, n = 8; IR + BMMC, n = 6. With ADP: SHAM, n = 9; IR, n = 9; IR + BMMC, n = 9. SEM: standard error of the mean; ANOVA: analysis of variance; IR: ischemia followed by reperfusion; BMMC: bone marrow mononuclear cells.

Quantification of the total Ca²⁺ load required for the return to baseline of fluorescence ([Ca²⁺]_t) and the time required for half-depolarization (t_1/2) is presented in Fig. 5. In IR mitochondria, 40% less [Ca²⁺]_t sufficed for total depolarization (Fig. 5A), which also took place with a t_1/2 that is 45% lower (Fig. 5B). Administration of BMMC totally normalized [Ca²⁺]_t, with a partial effect on t_1/2 (Fig. 5A, B). When assayed in the presence of ADP, [Ca²⁺]_t was similar in the three groups (Fig. 5C), with ADP-induced increases of 70%, 150%, and 70% in SHAM, IR, and IR + BMMC, respectively, compared with the values in the absence of the nucleotide (Fig. 5C vs A). The same profile was found for t_1/2, which also increased 60%, 160%, and 80% when one compares the same experimental group in the presence and absence of ADP (Fig. 5D vs B). Despite the small differences in the responses of [Ca²⁺]_t and t_1/2 to BMMC and ADP, the two parameters are closely associated in all conditions (Fig. 6): A single function was adjusted to the ensemble of different experimental points (P < 0.0001).

Figure 6.

Linear correlation between [Ca²⁺]_t and the corresponding t_1/2 of depolarization. Values are those taken from Fig. 5. Empty symbols: assays in the absence of ADP. Filled symbols: assays in the presence of 1 mM ADP. SHAM: empty and filled circles. IR: empty and filled triangles. IR + BMMC: empty and filled squares. The linear function t_1/2 = 22.1 µM^–1s × [Ca²⁺]_t was adjusted to the experimental values by least squares (P < 0.0001). IR: ischemia followed by reperfusion; BMMC: bone marrow mononuclear cells.

Stimulation of Citrate Synthase Activity by BMMC

The negative impact of IR in renal mitochondrial respiration and its recovery by BMMC has previously been demonstrated¹⁹. The following experiments investigated whether replenishing of the Krebs cycle with 2C fragments²⁵ is affected by IR and recovered by BMMC, as described for Ca²⁺ transport and Ca²⁺-induced modifications of mitochondrial membrane potential. Initially, activity was measured to investigate early effects at 1 h of reperfusion (Fig. 7A), as in the case of the other two processes above. Unexpectedly, the activity was similar in SHAM and IR groups, and 70% higher in IR + BMMC. We further investigated this result in experiments with a 24-h longer period of reperfusion (Fig. 7B), that showed the recovery of citrate synthase activity in SHAM and IR mitochondria to the levels in the group IR + BMMC, which remained similar to that seen after 1 h of reperfusion. This scenario is better seen in Fig. 8, which compares the activity in the three groups at two times: spontaneous and significant increase in SHAM and IR mitochondria with time, without modification in the case of IR + BMMC.

Figure 7.

BMMC stimulate citrate synthase activity. Citrate synthase activity was measured 1 h (A) and 24 h (B) after IR, as described in the “Materials and Methods” section. Groups are indicated on the abscissae. Bars represent means ± SEM. Differences were assessed with the use of one-way ANOVA followed by Tukey’s test, which are indicated within the panels. After 1 h: SHAM, n = 6; IR, n = 6; IR + BMMC, n = 9. After 24 h: SHAM, n = 5; IR, n = 7; IR + BMMC, n = 9. BMMC: bone marrow mononuclear cells; IR: ischemia followed by reperfusion; SEM: standard error of the mean; ANOVA: analysis of variance; TNB: 5-thio-2-nitrobenzoic acid.

Figure 8.

Evolution of citrate synthase activity between 1 and 24 h of interventions: (A) SHAM, (B) IR, and (C) IR + BMMC. Data and times are those described in the legend to Fig. 7 for the different groups and treatments. Bars represent means ± SEM. Differences between 1 and 24 h were assessed with the use of unpaired Student’s t test within each experimental group; the P values are indicated within the panels. IR: ischemia followed by reperfusion; BMMC: bone marrow mononuclear cells; SEM: standard error of the mean; TNB: 5-thio-2-nitrobenzoic acid.

Discussion

The main findings of this study were that BMMC recover the Ca²⁺ handling in renal cortical mitochondria isolated after IR, and that it ameliorated the response of mitochondrial membrane potential to depolarizing Ca²⁺ pulses. The immediate paracrine effects of BMMC, which result in important improvements in renal function^20–22, seem to rely on the preservation of the finely regulated mechanisms of Ca²⁺ handling, as demonstrated here. In previous studies, we have demonstrated that IR impairs mitochondrial respiration in both basal and phosphorylating conditions, as well as the generation of mitochondrial membrane potential¹⁹. The present data indicate that altered mechanisms for uptake and release of Ca²⁺ are key in processes that culminate in reduced ATP synthesis in IR. Ca²⁺ is an activator of dehydrogenases^29,30 and therefore of electron fluxes, generation of mitochondrial membrane potential, and ATP synthesis. Deregulation of Ca²⁺ transport mechanisms by IR in a tissular microenvironment with elevated formation of ROS^13,31 could facilitate the opening of PTP, as suggested by the experiments shown in Figs. 2 and 3.

These figures provide evidence that IR induced PTP opening, as suggested by the initially slow and then sudden tendency of a Ca²⁺ efflux (the smooth curve in Fig. 3A). We propose that PTP opening is prevented by BMMC because the effect is similar to that encountered in the presence of CsA, which reverses the IR-induced alterations in mitochondrial Ca²⁺ handling. Decrease in the extra-mitochondrial [Ca²⁺] after successive Ca²⁺ pulses (decrease in the first-order constant of the departure from the baseline of Calcium green fluorescence from 0.0051 to 0.0039 µM^–1 min^–1; see Fig. 2H), the recovery of the net rate of Ca²⁺ influx, and the demonstration that extra [Ca²⁺] (more 60%) that can be provided to the medium before the beginning of the Ca²⁺ efflux tendency in the presence of CsA (shaded area in Fig. 3B) give support to this view. CsA inhibits PTP opening³² and reduces cell death^33,34, which is a prominent event in renal IR¹⁹ mediated by PTP opening, as also demonstrated in heart lesions^13,35,36.

The target for the alterations by IR of the renal mitochondrial Ca²⁺ influx is possibly the mitochondrial Ca²⁺ uniporter (MCU)^37,38, its tetrameric structure preserved from fungi to humans^39,40 being destabilized by the intense production of ROS after IR. In a recent communication⁴¹, we demonstrated that intense oxidative stress is responsible for the early lesions caused by IR, which is avoided by administration of the main paracrine secretion of stem and mesenchymal cells (MSC)—the EVs they secrete⁴². BMMC, which contains a small parcel of MSC⁴³, has the same effect in preserving renal structures^{14,15,17,22,44}, demonstrating that they contain a population of cells that helps in the preservation of renal structure and function. Moreover, BMMC also secretes EVs, and blockade of EV secretion by GW4869 cancels the repair of an infarcted myocardium, as was recently demonstrated⁴⁵. As inhibition of catalase suppresses the beneficial effects of EVs⁴⁶, it is plausible that the effects of BMMC seen in Fig. 3 rely on the restoration of the normal redox environment, which is highly altered after IR in the kidney and heart^{13,22,47–49}. In the kidney, intravenous injection of BMMC is cytoprotective and has regenerative properties in a rat model of IR by modulating oxidative and inflammatory processes²².

CsA-sensitive PTP also modulates physiologically the mitochondrial membrane potential and local Ca²⁺ signals in mitochondria⁵⁰. However, long-lasting, deregulated PTP opening, together with exacerbated oxidative stress, is associated with several pathological processes in mitochondria, including the collapse of the mitochondrial membrane potential^51,52. It is plausible to associate the accelerated collapse of ΔΨ to alterations in IR-induced Ca²⁺ handling for two reasons. First, IR decreased the tightly associated (Fig. 6) [Ca²⁺]_t and the t_1/2 required for Ca²⁺-induced depolarization (Figs. 4 and 5A, B). Second, the influence of IR was not detected in both parameters in the presence of ADP (Fig. 5C, D), probably because the nucleotide decreases the opening of PTP⁵³ through a mechanism that seems to involve an increase in the K_m of Ca²⁺-induced PTP opening, as proposed by Bauer and Murphy¹³. Stabilization of adenine nucleotide translocase (ANT) by ADP in a conformation facing the matrix inhibits PTP opening³⁵, which could explain why the influence of IR in [Ca²⁺]_t and t_1/2 is not seen when ADP is present in the assays.

The influence of ADP on the Ca²⁺-induced depolarization in IR opens up the possibility of an additional target for the injury and for the beneficial effect of BMMC administration, namely, the transport of Ca²⁺ across the outer mitochondrial membrane (OMM). Ca²⁺ uptake across the OMM is mediated and regulated by voltage-dependent anion channels (VDACs) that form large Ca²⁺ pores^54,55, which are modulated by interactions between the channel and ANT. Allouche et al⁵⁶ demonstrated the existence of a direct VDAC-ANT interaction and, several years before^57,58 (cf. Bauer and Murphy¹³), it was proposed that they form a complex in physiological and pathological conditions (including IR). Thus, this complex could participate in PTP opening as a consequence of IR, making it possible that BMMC secrete factors (including EV) that restore the architecture and function of the VDAC-ANT complex together with those of MCU.

The generation of the mitochondrial membrane potential and its utilization to synthesize ATP by the F_oF₁-ATP synthase requires an appropriate seeding of the Krebs cycle and the mitochondrial electron transport. The citrate synthase is the key enzyme at the beginning of the cycle that ensures the continuous supply of 2 C atoms²⁵, and for this reason we investigated whether it was affected by IR and recovered or preserved by BMMC. One hour after the recovery of reperfusion (1.5 h after the beginning of the surgery), the SHAM and IR levels (Fig. 7A) were similar and, possibly, this was a consequence of the renal pedicle manipulation, without any additional influence of arterial clamping and PTP opening. Possibly because the impact on the enzyme was mild and independent of mitochondrial Ca²⁺ disturbance, activity in the SHAM and IR rats increased in the following 24 h, reaching the levels encountered in the group IR + BMMC 1 h after the acute lesion (Fig. 7B). Thus, it may be that the IR + BMMC levels are the “true” normal values, preserved by BMMC administration. Manipulation of the renal pedicle in SHAM mice provokes a specific transcriptional response of vimentin, a marker of renal lesion⁵⁹, which involves the sympathetic nervous system and the local renin–angiotensin system that are also crucial for the pathogenesis of renal lesions in IR⁶⁰.

In conclusion, we have demonstrated that BMMC prevent renal mitochondrial lesions during the AKI caused by IR through mechanisms associated with the preservation of Ca²⁺ influx and release, and with the response of ΔΨ to Ca²⁺. It is possible that the preservation of these mechanisms relies—at least in part—on the control of the microenvironmental production of ROS⁴¹ in early periods after IR.

Footnotes

Acknowledgements

The authors are grateful to Glória Costa-Sarmento and Rosilane Taveira for their excellent technical assistance. The finally submitted version of the manuscript was prepared by BioMedES Ltd, UK ().

Ethical Approval

All experimental protocols followed the guidelines of the Committee of Ethics in the Use of Animals for Research at Federal University of Rio de Janeiro, Brazil (approval number: 104).

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article, and informed consent is not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the Brazilian National Research Council (CNPq; grants 470266/2014-7 and 401816/2016-8), the Carlos Chagas Rio de Janeiro State Foundation (FAPERJ; grants E-26/111.633/2014, E-26/201.736/2015, E-26/202.963/2017, and E-26/201.558/2018), the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES; grants 88887.124150/2014-00 and 88887.320213/2019-00), and National Institute of Science and Technology in Regenerative Medicine (REGENERA; grant 465656/2014-5).

ORCID iD

Adalberto Vieyra

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Bone Marrow Mononuclear Cells Restore Normal Mitochondrial Ca 2+ Handling and Ca 2+ -Induced Depolarization of the Internal Mitochondrial Membrane by Inhibiting the Permeability Transition Pore After Ischemia/Reperfusion

Abstract

Keywords

Introduction

Materials and Methods

Animals

Cells

Mitochondrial Isolation

Mitochondrial Ca2+ Accumulation and Release

Ca2+-Induced Depolarization of the Inner Mitochondrial Membrane

Citrate Synthase Activity

Statistical Analysis

Results

IR Decreased Rapid Ca2+ Uptake by Mitochondria From Proximal Tubule Cells: Total Recovery by BMMC

IR Alters the Response to Ca2+ of Mitochondrial Membrane Potential: Effect of BMMC and ADP

Stimulation of Citrate Synthase Activity by BMMC

Discussion

Footnotes

Acknowledgements

Ethical Approval

Statement of Human and Animal Rights

Statement of Informed Consent

Declaration of Conflicting Interests

Funding

ORCID iD

References

Mitochondrial Ca²⁺ Accumulation and Release

Ca²⁺-Induced Depolarization of the Inner Mitochondrial Membrane

IR Decreased Rapid Ca²⁺ Uptake by Mitochondria From Proximal Tubule Cells: Total Recovery by BMMC

IR Alters the Response to Ca²⁺ of Mitochondrial Membrane Potential: Effect of BMMC and ADP