Mitochondrial permeability transition pore opening induces the initial process of renal calcium crystallization

Free Radical Biology & Medicine 52 (2012) 1207–1217

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Original Contribution

Mitochondrial permeability transition pore opening induces the initial process of renal calcium crystallization Kazuhiro Niimi a, Takahiro Yasui a,⁎, Masahito Hirose b, Shuzo Hamamoto a, Yasunori Itoh c, Atsushi Okada a, Yasue Kubota a, Yoshiyuki Kojima a, Keiichi Tozawa a, Shoichi Sasaki a, Yutaro Hayashi a, Kenjiro Kohri a a b c

Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan Department of Urology, Kainan Hospital, Aichi Prefectural Welfare Federation of Agricultural Cooperatives, Yatomi, Japan Department of Urology, Nagoya City West Medical Center, Nagoya, Japan

a r t i c l e

i n f o

Article history: Received 31 August 2011 Revised 27 December 2011 Accepted 9 January 2012 Available online 18 January 2012 Keywords: Mitochondrial permeability transition pore Renal calcium crystallization Cyclosporine A Oxidative stress Renal tubular cell injury Free radicals

a b s t r a c t Renal tubular cell injury induced by oxidative stress via mitochondrial collapse is thought to be the initial process of renal calcium crystallization. Mitochondrial collapse is generally caused by mitochondrial permeability transition pore (mPTP) opening, which can be blocked by cyclosporine A (CsA). Definitive evidence for the involvement of mPTP opening in the initial process of renal calcium crystallization, however, is lacking. In this study, we examined the physiological role of mPTP opening in renal calcium crystallization in vitro and in vivo. In the in vitro study, cultured renal tubular cells were exposed to calcium oxalate monohydrate (COM) crystals and treated with CsA (2 μM). COM crystals induced depolarization of the mitochondrial membrane potential and generated oxidative stress as evaluated by Cu-Zn SOD and 4-HNE. Furthermore, the expression of cytochrome c and cleaved caspase 3 was increased and these effects were prevented by CsA. In the in vivo study, Sprague–Dawley rats were administered 1% ethylene glycol (EG) to generate a rat kidney stone model and then treated with CsA (2.5, 5.0, and 10.0 mg/kg/day) for 14 days. EG administration induced renal calcium crystallization, which was prevented by CsA. Mitochondrial collapse was demonstrated by transmission electron microscopy, and oxidative stress was evaluated by measuring Cu-Zn SOD, MDA, and 8-OHdG generated by EG administration, all of which were prevented by CsA. Collectively, our results provide compelling evidence for a role of mPTP opening and its associated mitochondrial collapse, oxidative stress, and activation of the apoptotic pathway in the initial process of renal calcium crystallization. © 2012 Elsevier Inc. All rights reserved.

Environmental and genetic factors are responsible for causing kidney stone disease. However, efficient methods of prophylaxis have not been established despite the increasing prevalence of the disease, mainly because the mechanism of stone formation has not been described in detail [1]. Renal tubular cell injury is regarded as a major risk factor for renal calcium crystallization [2,3], which can be prevented by antioxidants such as citric acid, vitamin E, traditional medicinal herbs, and green tea [4–7]. Cell membrane injury caused by oxidative stress induces the attachment of crystals to renal tubular cells [8–10], which express osteopontin (OPN), a major component of stone matrix protein [11,12]. Its expression and structure could affect experimental renal

Abbreviations: COM, calcium oxalate monohydrate; CsA, cyclosporine A; EG, ethylene glycol; 4-HNE, 4-hydroxy-2-nonenal; mPTP, mitochondrial permeability transition pore; MDA, malondialdehyde; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; OPN, osteopontin; PLOM, polarized light optical microphotography; ROS, reactive oxygen species; SOD, superoxide dismutase; TEM, transmission electron microscopy; TMRE, tetramethylrhodamine ethyl ester. ⁎ Corresponding author. Fax: + 81 52 852 3179. E-mail address: [email protected] (T. Yasui). 0891-5849/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2012.01.005

calcium crystallization [13]. Many current models of calcium oxalate stone formation suggest that the generation of reactive oxygen species (ROS) stored within mitochondria is intimately associated with renal tubular cell injury [14] and the process of renal calcium crystallization [15–17]. Our recent study also indicated that oxidative stress caused by mitochondrial collapse is involved in the early phase of renal calcium crystallization in mice [18]. Mitochondrial collapse is generated by mitochondrial permeability transition pore (mPTP) opening, which plays an important role in the mechanism of cell death through mitochondrial dysfunction. These pores consist of cyclophilin D, which penetrates the inner and outer membranes of mitochondria [19,20]. When the mPTP opens, cytosolic protons (H +) flow into the mitochondrial matrix and disrupt the membrane potential, causing the swelling and subsequent collapse of mitochondria [21]. The opening of the mPTP depends on the activation of cyclophilin D located in the mitochondrial matrix. When cyclophilin D remains inactive, the mPTP does not open and the mitochondrion remains intact; thus, inactivating cyclophilin D can prevent mitochondrial collapse [22]. Recent reports showed that cyclosporine A (CsA) [23] can block mPTP opening via inactivation of cyclophilin D.

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In this study, we have examined the changes in mitochondrial membrane potential and oxidative stress in an in vitro study; additionally, we evaluated mitochondrial structure and renal calcium crystallization in an in vivo study. We demonstrate that depolarization of the mitochondrial membrane potential and oxidative stress were prevented by CsA. We further show that mitochondrial collapse and renal calcium crystallization were significantly decreased with CsA treatment. Together, these results provide compelling evidence for a role of mPTP opening and the associated mitochondrial collapse, oxidative stress, and activation of the apoptotic pathway in the initial process of renal calcium crystallization. Materials and methods Preparation of calcium oxalate monohydrate (COM) crystal suspensions Oxalic acid (200 mM, 0.5 ml) and 200 mM calcium chloride were mixed at room temperature to a final concentration of 10 mM, and the COM crystals that immediately formed in suspension were equilibrated for 3 days. The COM crystals were then washed three times with sodium and chloride-free distilled water saturated with calcium oxalate, resuspended to a final concentration of 2.92 mg/ml, and adjusted to pH 6.8 [24]. Cell culture The renal proximal tubular cell line NRK-52E (American Type Culture Collection, Rockville, MD, USA) was cultured in Dulbecco's modified Eagle's medium supplemented with 8% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Gibco-BRL, Rockville, MD, USA). The cells were routinely seeded at a density of 1 × 10 5/60-mm culture dish (Nalge Nunc, Naperville, IL, USA) at 37 °C in a humidified atmosphere of 5% CO2 in air. The medium was changed every third day and the cells were subcultured before forming confluent monolayers. NRK-52E cells were seeded at a density of 1 × 10 6/90-mm dish and cultured to 90% confluence. The cells were then treated with or without CsA (2 μM) for 10 min and then with COM crystals (100 μg/cm 2). Measurement of mitochondrial membrane potential (Δψm) Cells were loaded with the membrane potential-sensitive dye tetramethylrhodamine ethyl ester perchlorate (TMRE; 20 nM in Hepes-buffered salt solution; Invitrogen, Carlsbad, CA, USA) [25]. Cells loaded with TMRE were then analyzed using a confocal microscope (LSM5 PASCAL; Carl Zeiss Co. Ltd., Oberkochen, Germany) equipped with ×20 and ×100 oil-immersion, quartz objective lenses. The cells were then treated with or without CsA (2 μM) for 10 min and then with COM crystals (100 μg/cm 2) for 0, 5, 10, 15, and 30 min. As a negative control, untreated NRK-52E cells were observed, and as a positive control, we used carbonyl cyanide mchlorophenyl hydrazone (CCCP; 10 μM), an uncoupler that causes mitochondrial depolarization [26]. Mitochondrial fidelity in cells stained with TMRE was quantified by flow cytometry. After three washes with phosphate-buffered saline (PBS) to remove COM crystals, stained cells in each group were detached using 0.05% trypsin– EDTA, washed with PBS, and diluted to 1 ml. A total of 30,000 events were collected from each sample and the data were displayed on a logarithmic scale of increasing red fluorescence intensity using a FACSCalibur HG (Becton–Dickinson, Franklin Lakes, NJ, USA). Isolation of mitochondria and cytosol Mitochondria and cytosol were isolated from NRK-52E cells using the Mitochondria Isolation Kit for Cultured Cells (Pierce Biotechnology, Rockford, IL, USA) [27]. Briefly, after Dounce homogenization, lysates

were centrifuged at 700 g for 10 min to precipitate nuclei and garbage. The supernatants were then centrifuged at 12,000 g for 15 min; the pellet contained the isolated mitochondria and the supernatant contained the cytosol fraction. The cytosol fraction was used for Western blotting to detect cytochrome c. Western blotting NRK-52E cells stored at −20 °C were immersed in 1× lysis buffer and lysed by sonication on ice. The total protein concentration in the supernatant was spectrophotometrically quantified using an Ultrospec 3100 Pro (GE Healthcare, Wallingford, CT, USA). Samples containing 30 μg total protein were mixed with loading buffer (Laemmli sample buffer; Bio-Rad Laboratories, Hercules, CA, USA). Proteins were resolved by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto Immobilon polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Blocking for 1 h at room temperature was followed by an overnight incubation with a polyclonal anti-rat superoxide dismutase (SOD) antibody (dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse 4-hydroxy-2-nonenal (4-HNE) monoclonal antibody (dilution 1:4; Nikken Seil, Shizuoka, Japan), and anti-rat cleaved caspase 3 antibody (dilution 1:1000; Cell Signaling Technology, Beverly, MA, USA) at 4 °C. After being washed, the membranes were treated with the corresponding peroxidase-conjugated secondary antibodies for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence Western blotting analysis kits (Pierce Biotechnology). The membranes were probed with a β-actin antibody as a loading control (Sigma–Aldrich, St. Louis, MO, USA). For Western blotting of cytochrome c, a similar procedure was followed except for the use of samples (cytosol fraction isolated from NRK-52E cells) containing 10 μg total protein and anti-rat cytochrome c antibody (dilution 1:1000; Cell Signaling Technology). The same membrane was used for each CsA (−) COM (+) and CsA (+) COM (+) group to ensure uniformity under the conditions. The protein expression levels in the bands corresponding to SOD, 4-HNE, cytochrome c, and cleaved caspase 3 (n = 5 each) were quantified using Image Quant LAS 4000 (GE Healthcare Japan, Tokyo, Japan), which is a multipurpose CCD camera system for quantitative imaging of blots developed by Amersham for enhanced chemiluminescence, with standard UV transillumination for ethidium bromide gel visualization. Experimental animals All experiments proceeded with the approval of the Animal Care Committee of the Faculty of Medicine, Nagoya City University Graduate School of Medical Sciences. Male Sprague–Dawley (SD) rats (Charles River Japan, Yokohama, Japan), age 7 weeks and weighing 280–320 g, were acclimated at 23 ± 1 °C on a 12-h light/ dark cycle for 7 days in metabolic cages before experiments were started. All animals had free access to standard rat food (containing calcium, 1.12 g; phosphorus, 0.9 g; magnesium, 0.26 g; and sodium, 0.21 g/100 g; Oriental Yeast Co., Tokyo, Japan). Hyperoxaluric rat model and cyclosporine A administration Forty-eight SD rats were given free access to water containing 1% ethylene glycol (EG) to form stones [28] and then treated two times per day with CsA via a gastric tube. The rats were assigned to one of the following groups (n = 12 per group) and weighed weekly: one group received EG only (EG group) and three EG and CsA groups also received 2.5, 5, or 10 mg/kg/day CsA (EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0 groups, respectively). At 7 and 14 days after the start of drug therapy, blood was sampled from the inferior vena cava of 6 rats per group. These rats were sacrificed under ether

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anesthesia and both kidneys were immediately excised and dissected. One kidney was histologically examined and RNA was extracted from the other. Two days before euthanasia, the rats were placed individually in metabolic cages for 24 h and urine samples were collected into cups containing HCl for oxalate measurements. The kidneys and urine samples were obtained from control rats without EG or CsA at day 0 (n = 6).

Mann–Whitney U test. A P value of b0.05 denotes a statistically significant difference.

Detection of renal calcium crystallization

Fig. 1a shows NRK-52E cells stained with TMRE. Red fluorescent bodies indicate TMRE accumulation in mitochondria, which are distributed around nuclei stained blue with Hoechst 33258. The negative control cells exhibited no change in TMRE intensity throughout the experimental period. In non-CsA-treated cells exposed to COM crystals (CsA (−) COM (+)), the TMRE intensity gradually diminished between 5 and 30 min. In contrast, TMRE intensity in CsA-treated cells exposed to COM crystals (CsA (+) COM (+)) remained nearly the same from 5 to 30 min. In the positive control (CCCP), TMRE intensity almost disappeared within 5 min after the addition of CCCP and completely disappeared by 30 min. The nuclear dye did not change throughout the experimental period. There was a clear difference between the groups. Fig. 1b shows the results of flow cytometry of NRK-52E cells stained with TMRE. In CsA (−) COM (+) cells, the peak TMRE intensity was shifted to the left by 15 min and was shifted slightly more to the left by 30 min. However, in CsA (+) COM (+) cells, the shift in peak TMRE intensity was negligible at 15 and 30 min.

Renal specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Cross sections (4 μm thick) were stained using the Pizzolato method to detect crystals containing oxalate. Briefly, dewaxed paraffin sections were rinsed with distilled water and then a mixture of 1 ml each of 30% hydrogen peroxide and 5% silver nitrate (pH 6.0) was poured onto the tissue sections. The sections were placed 15 cm from an incandescent 60-W lamp for 15–30 min. After gas bubbles developed, the solution was poured off and replaced with a fresh hydrogen peroxide/silver nitrate mixture. The sections were thoroughly rinsed with distilled water, counterstained with Kernechtrot (nuclear fast red) solution (Merck KGaA, Darmstadt, Germany), and then dehydrated in a graded ethanol series. The amount of crystals was determined as previously described [29]. Briefly, dewaxed unstained 4-μm-thick cross sections were examined using polarized light optical microphotography (BX51-33P-O; Olympus, Tokyo, Japan). Images of sections were scanned and regions with crystals with strong birefringence were measured and expressed as ratios (%) of the total tissue area of kidney cross sections by using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA). Immunohistochemical staining Oxidative stress and renal tubular cell injury were estimated by immunohistochemical staining for SOD, malondialdehyde (MDA), 8-hydroxy-2′-deoxyguanosine (8-OHdG), and OPN. Serial sections (4 μm thick) were microwave-heated for 15 min, and then endogenous peroxidase activity was blocked with 0.5% hydrogen peroxide in methanol for 30 min. The sections were washed in 0.01 M PBS and then nonspecific binding was blocked by incubation in PBS containing skim milk for 1 h at room temperature. The sections were then treated with anti-rat SOD, MDA, 8-OHdG, and OPN antibodies (SOD, MDA, OPN from Santa Cruz Biotechnology, 8-OHdG from Nikken Seil) overnight at 4 °C. Positive reactions were detected using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. Images of sections were scanned and the protein expression areas for SOD, MDA, 8-OHdG, and OPN were measured for 20 kidneys and expressed as ratios (%) of the total tissue area of kidney cross sections using Image-Pro Plus software (Media Cybernetics). Transmission electron microscopy (TEM) The microstructure of mitochondria in the kidney was examined using TEM. Kidneys were perfusion-fixed using 20 ml of 0.1 M phosphoric acid buffer and 20 ml of 2.5% glutaraldehyde, extracted and washed with phosphoric acid buffer, and then fixed with 2% osmium tetraoxide for 2 h. Tissues were dehydrated using a graded series of ethanol (50–100%), embedded in epoxy resin, and polymerized at 60 °C for 48 h. Super slices (99 nm) were double stained with uranium and lead for observation using a JEM-1011 TEM microscope (JEOL Ltd., Tokyo, Japan). Statistical analysis All data are expressed as means ± standard deviation. The statistical significance of differences among groups was examined using the

Results Changes in mitochondrial membrane potential in NRK-52E cells induced by COM

SOD, 4-HNE, cytochrome c, and cleaved caspase 3 expression changes in NRK-52E cells exposed to COM Figs. 2a and b show Western blot detection of time-dependent change for SOD and 4-HNE. In CsA (−) COM (+) cells, the expression of SOD showed a decrease from 1 to 6 h after exposure to COM crystals, whereas no significant changes were detected in CsA (+) COM (+) cells. There were significant differences between CsA (−) COM (+) and CsA (+) COM (+) at 1, 3, and 6 h after exposure to COM crystals. On the other hand, the expression of 4-HNE increased in correlation with exposure to COM crystals up to 6 h in CsA (−) COM (+) cells, whereas only a moderate increase was noted in CsA (+) COM (+) cells. There were significant differences between CsA (−) COM (+) and CsA (+) COM (+) at 15 min, 1 h, and 3 h after exposure to COM crystals. Figs. 3a and b show Western blot detection of time-dependent change for cytosolic cytochrome c and cleaved caspase 3. In CsA (+) COM (+) cells, the expression of cytosolic cytochrome c was almost undetectable 1 h after exposure to COM crystals and then increased by 3 and 6 h, although the expression was lower than that in CsA (−) COM (+) cells. There were significant differences between CsA (−) COM (+) and CsA (+) COM (+) at 1, 3, and 6 h after exposure to COM crystals. In CsA (+) COM (+) cells, the expression of cleaved caspase 3 increased 1, 3, and 6 h after exposure to COM crystals, but the expression was lower than that in CsA (−) COM (+) cells. There were significant differences between CsA (−) COM (+) and CsA (+) COM (+) at 1, 3, and 6 h after exposure to COM crystals. Calcium crystal deposits in rats administered ethylene glycol Fig. 4a shows the findings of Pizzolato staining and polarized light optical microphotography (PLOM). Calcium oxalate was deposited in the rat kidneys (renal calcium crystallization) 14 days after EG administration. Pizzolato-stained brown regions indicate calcium oxalate deposits detected in the renal tubules, mainly located at the corticomedullary junction in the EG group. The crystals appeared to attach to the apical sides of renal tubular cells. In contrast, calcium oxalate deposits were sparse and minimal in the renal tubular cells of

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a

0 min

5 min

10 min

15 min

30 min

Negative Control

CsA(-) COM(+)

CsA(+) COM(+)

CCCP

b

CsA(-) COM(+)

CsA(+) COM(+)

0 min 15 min 30 min Fig. 1. Mitochondrial membrane potential in NRK-52E cells. (a) Confocal laser scanning microscopy images of NRK-52E cells stained with tetramethylrhodamine ethyl ester perchlorate (TMRE). Blue (Hoechst 33258)-stained structures are nuclei. Red-fluorescent bodies indicate TMRE accumulation in the mitochondria, which are distributed around the nuclei. Negative control: untreated NRK-52E cells; CsA (−) COM (+), non-CsA-treated cells exposed to COM crystals; CsA (+) COM (+), CsA (2 μM)-treated cells exposed to COM crystals; CCCP, cells treated with carbonyl cyanide m-chlorophenyl hydrazone (10 μM). Original magnification, × 400; inset, × 1200. (b) Flow cytometry of NRK-52E cells stained with TMRE and changes in intensity over a 30-min period after the start of the experiment. CsA (−) COM (+) and CsA (+) COM (+) indicate the same as in (a). The vertical and horizontal axes indicate cell counts and TMRE intensity, respectively.

whole kidneys in the EG + CsA groups. The PLOM results are from the same field as the Pizzolato staining in both the EG and the EG + CsA groups. Fig. 4b shows the amount of calcium oxalate deposits in the each group. The crystals rapidly increased after 7 and 14 days in the EG group (0.085 ± 0.039% and 0.994 ± 0.287%, respectively), but not in the EG + CsA2.5 (0.006 ± 0.002% and 0.018 ± 0.004%, respectively), EG + CsA5.0 (0.002 ± 0.001% and 0.019 ± 0.013%, respectively), and EG + CsA10.0 (0.000 ± 0.000% and 0.002 ± 0.001%, respectively)

groups. Differences were significant between the EG and the EG + CsA groups, but there were no significant differences among the EG + CsA groups. Urinary and serum variables Table 1 shows urinary and serum variables. There were no changes in urine pH on days 7 and 14 after EG administration. In the EG group, urine volume was significantly lower than in the

K. Niimi et al. / Free Radical Biology & Medicine 52 (2012) 1207–1217

a

CsA(-) COM(+)

1211

CsA(+) COM(+)

SOD

4-HNE

-actin control 15 min 30 min

b

1h

3h

6h

SOD

* 1.0

*

*

3h

6h

4-HNE

10

1.2

1h

control 15 min 30 min

8

0.8 6

CsA (-) COM (+)

0.6

CsA (+) COM (+)

4 0.4

*

2

0.2

*

*

1h

3h

0

0 control 15min 30min

1h

3h

control 15min 30min

6h

6h

Fig. 2. Detection of SOD and 4-HNE by Western blotting. (a) Expression of SOD and 4-HNE. The bottom shows the expression of β-actin (42 kDa) used as an internal control. CsA (−) COM (+) and CsA (+) COM (+) indicate the same as Fig. 1a. (b) Expression of SOD and 4-HNE was quantified and expressed as rate of change (%) over time. The vertical axis indicates the rate of change (%) with respect to the control value. The horizontal axis indicates the time after exposure to COM crystals. *Statistically significant difference, CsA (+) COM (+) vs CsA (−) COM (+).

a

CsA(-) COM(+)

CsA(+) COM(+)

cytochrome c

cleaved caspase 3

actin control 1h

b

3h

6h

3h

control 1h

cytochrome c

6h

cleaved caspase 3

20

20

15

15

* CsA (-) COM (+)

10

10

* 5

*

* 5

*

CsA (+) COM (+)

*

0

0 control

1h

3h

6h

control

1h

3h

6h

Fig. 3. Detection of cytochrome c and cleaved caspase 3 by Western blotting. (a) Expression of cytosolic cytochrome c and cleaved caspase 3. The bottom shows the expression of β-actin (42 kDa) used as the internal control. CsA (−) COM (+) and CsA (+) COM (+) indicate the same as in Fig. 1a. (b) Expression of cytochrome c and cleaved caspase 3 was quantified and expressed as the rate of change (%) over time. The vertical axis indicates the rate of change (%) with respect to the control. The horizontal axis indicates the time after exposure to COM crystals. *Statistically significant difference, CsA (+) COM (+) vs CsA (−) COM (+).

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EG

EG+CsA5.0

EG+CsA2.5

EG+CsA10.0

c

Pizzolato

a

J

PLOM

P

M

b

% 1.4

1.2

1.0

0.8

EG EG+CsA2.5

0.6

EG+CsA5.0 0.4

EG+CsA10.0

0.2

* * *

* * *

0.0 Day 0

Day 7

Day 14

Fig. 4. Renal calcium crystallization. (a) Renal calcium crystallization was evaluated by Pizzolato staining and polarized light optical microphotography (PLOM) in rat kidneys on day 14. EG, EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0 represent rats administered EG or EG and cyclosporine A at 2.5, 5.0, and 10.0 mg/kg/day, respectively. P, papilla; M, medulla; J, corticomedullary junction; C, cortex. Pizzolato-stained brown regions indicate calcium oxalate deposits detected in the renal tubules. Original magnification, × 20; inset, × 400. (b) Quantification of renal calcium crystallization expressed as the ratio (%) of total cross-sectional tissue areas. *Statistically significant difference, EG vs EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0.

EG + CsA10.0 group. In the 24-h urine oxalate excretion measurement, there were significant differences between the control and the EG group on days 7 and 14, with a more than about 7.2- and 7.6-fold higher value in the EG group on days 7 and 14, respectively. On the other hand, there were no significant differences in 24-h

urine oxalate excretion between the EG group and the EG + CsA groups. There were no significant differences in serum calcium levels among the control, EG, and EG + CsA groups. Serum creatinine levels were similar to serum calcium levels, showing no significant differences among all groups.

Table 1 Urinary and serum variables in control and experimental rats. Group (day)

Control 7 14 EG 7 14 EG + CsA2.5 7 14 EG + CsA5.0 7 14 EG + CsA10.0 7 14

Volume (ml)

pH

Urine (mg/day)

Serum (mg/dl)

Oxalate

Calcium

Creatinine

Calcium

16.8 ± 8.6 16.1 ± 4.5

8.0 ± 0.0 8.1 ± 0.1

1.8 ± 0.2 1.7 ± 0.4

3.23 ± 1.21 3.32 ± 2.56

0.21 ± 0.03 0.21 ± 0.08

10.35 ± 0.38 10.85 ± 0.78

15.6 ± 6.4 15.1 ± 6.9#

8.1 ± 0.1 8.4 ± 0.5

12.9 ± 1.3* 13.0 ± 0.9*

3.95 ± 0.75 3.66 ± 1.00

0.27 ± 0.06 0.34 ± 0.16

10.83 ± 0.18 10.08 ± 0.08

20.3 ± 1.9 20.7 ± 4.8

8.4 ± 0.4 8.1 ± 0.1

10.2 ± 1.3* 14.8 ± 1.0*

3.09 ± 0.66 3.55 ± 0.71

0.25 ± 0.06 0.26 ± 0.02

10.65 ± 0.36 10.50 ± 0.32

23.7 ± 6.3 23.8 ± 13.3

8.0 ± 0.0 7.6 ± 1.5

10.8 ± 2.0* 14.1 ± 2.4*

4.39 ± 4.04 4.29 ± 2.05

0.20 ± 0.01 0.24 ± 0.06

10.30 ± 0.18 10.36 ± 0.52

18.2 ± 1.5 25.5 ± 9.5#

8.1 ± 0.2 8.3 ± 0.4

11.4 ± 1.0* 13.8 ± 2.1*

4.40 ± 1.10 3.90 ± 1.90

0.19 ± 0.02 0.22 ± 0.02

10.43 ± 0.24 10.58 ± 0.16

*P b 0.05, control vs EG, EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0. # P b 0.05, EG vs EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0.

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Immunohistochemical staining for SOD, MDA, 8-OHdG, and OPN in the rat kidneys Fig. 5 shows the results of immunohistochemical staining in the rat kidneys on day 14 for SOD, MDA, 8-OHdG, and OPN. All the images represent serial sections. SOD expression is shown in the cortex, and MDA, 8-OHdG, and OPN expression is shown in the corticomedullary junction. In the control group, renal tubular cells in the cortex expressed higher levels of SOD than those in the EG group. The expression of SOD was similar between the EG + CsA (EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0) and the control groups, and there were no significant differences among the three EG + CsA groups. Furthermore, in the EG group, the ratio of the SOD expression area was significantly lower than those in the other groups. In the control group, MDA production was undetectable in the renal tubular cells of whole kidneys, but MDA was detectable in

a

EG

some renal tubular cells around the corticomedullary junction of the EG group kidneys. In the EG + CsA groups, MDA production was undetectable. In the EG group, the ratio of the MDA expression area was significantly higher than those in the other groups. In the control group, the expression of 8-OHdG was undetectable in the renal tubular cells of whole kidneys, but in the EG group, the expression was detectable in the nuclei of renal tubular cells of whole kidneys. In the EG + CsA groups, the expression of 8-OHdG was detectable in some renal tubular cells around the corticomedullary junction. In the EG group, the ratio of the 8-OHdG expression area was significantly higher than those in the other groups. In the control group, the expression of OPN was barely detectable, but in the EG group, it was evenly and densely distributed throughout the renal tubular cells of whole kidneys. OPN expression was detected in some renal tubular cells in each of the EG + CsA groups. In the EG group, the ratio of the OPN expression area was significantly higher than those in the other groups.

EG+CsA2.5

EG+CsA5.0

EG+CsA10.0

OPN

8-OHdG

MDA

SOD

Control

1213

b

% 50

#

#

#

40

% 2.0

*

1.5

30 1.0 20 10

#

*

0

% 20

0.5 0.0

SOD

#

# MDA

% 20

*

*

15

15

10

10

control EG

5 0

# 8OHdG

#

#

5 0

EG+CsA2.5

#

#

EG+CsA5.0

#

EG+CsA10.0

OPN

Fig. 5. (a) Immunohistochemical detection of superoxide dismutase (SOD), malondialdehyde (MDA), 8-hydroxy-2′-deoxyguanosine (8-OHdG), and osteopontin (OPN) on day 14. EG, EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0 represent rats administered with EG or EG and cyclosporine A at 2.5, 5.0, and 10.0 mg/kg/day, respectively. Original magnification, × 200. (b) The graphs represent the ratio of the area of expression of SOD, MDA, 8-OHdG, or OPN to the total area of kidney cross sections. The vertical axis indicates the ratio of expression area. *Statistically significant difference, control vs EG. #Statistically significant difference, EG vs EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0.

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Control

EG

EG+CsA2.5 N

N

S S

S

LU

N

MV

S

MV

MV

LU

LU N

S

MIT

MIT

S

EG+CsA5.0

N

EG+CsA10.0

N

MV N

MV

LU

N MIT

MIT

LU

MIT

Fig. 6. Ultrastructural observation by transmission electron microscopy (TEM) on day 14. The ultrastructural details of mitochondria in renal tubular cells as observed by TEM in control, EG, and EG + CsA groups are shown. Scale bars, 10 μm. N, nuclei; MV, microvilli; LU, lumen; MIT, mitochondria; S, trace of stone. The marked electron-dense rings around the tubule represent hyalinization.

Ultrastructural findings of rat kidneys exposed to EG Fig. 6 shows the ultrastructure of rat kidney sections examined by TEM at day 14. The renal tubules were circular, microvilli were evident in the lumen, and mitochondria were located around the nuclei in the control group. In contrast, renal tubules of the EG group were thin with flattened tubular cells, the lumen of the renal tubule was expanded, microvilli were barely recognizable, and crystals were present in the lumen. Swollen mitochondria resembling fat droplets around the nuclei had an indistinct, discontinuous, and partly collapsed double membrane. However, the renal tubules of the EG + CsA (EG + CsA2.5, EG + CsA5.0, and EG + CsA10.0) groups were circular and microvilli were detected in the lumen; they were longer than those in the EG group, and the layer was thicker, but slightly shorter than that in the control group. The mitochondria had a regular internal structure with a continuous double membrane, similar to the mitochondria in the control group. Discussion The mechanism of renal calcium crystallization remains unclear because kidney stone pathogenesis is multifactorial. Renal tubular cell injury affects the initial process of renal calcium crystallization through unknown mechanisms [3]. Inflammation and intracellular morphological changes via oxidative stress are considered essential to renal calcium crystallization. Studies have shown that in renal tubular cells exposed to COM crystals, the generation of ROS is induced via mitochondrial collapse. Mitochondria are major sources of intracellular ROS because they are sites of aerobic metabolism, and mitochondrial ROS production is increased under conditions of cell damage or increased stress [30]. COM crystals stimulate renal tubular cells to generate superoxide (O2•−) via NADPH oxidase [31]. Furthermore, O2•− activates cyclophilin D, which leads to mPTP opening [19]. Therefore, exposure to COM crystals is considered to induce mitochondrial collapse. On the other

hand, CsA, which is considered an mPTP inhibitor, blocks mPTP opening and can therefore prevent mitochondrial collapse. The opening of the mPTP changes the Δψm, which can be monitored using TMRE, a fluorescent probe that accumulates in polarized mitochondria and is released upon their depolarization. Cytochrome c is an important protein that induces apoptosis, and it is released into the cytosol upon mitochondrial collapse. Binding of the released cytochrome c to cytosolic apoptosis protease activating factor 1 activates caspase 9 and caspase 3, which results in the induction of apoptosis [32]. During this process, ROS are also released from the intramembrane compartment into the cytosol [30], which further injures renal tubular cells. In our in vitro study, the disappearance of TMRE indicated mitochondrial collapse through depolarization of the mitochondrial membrane (Fig. 1). Disappearance of TMRE was gradual after exposure to COM crystals. We hypothesize that mitochondrial injury by COM crystals preceded a change in gene expression in the renal tubular cells. Decreased SOD expression [33] and increased 4-HNE expression indicated an increase in oxidative stress in renal tubular cells. Increased cytosolic cytochrome c expression and cleaved caspase 3 expression were indicative of mitochondrial collapse and the release of cytochrome c from the mitochondrial membrane into the cytosol through induction of the apoptotic pathway [34] by COM crystals (Fig. 2). The ability of CsA to diminish these changes was also shown. In the in vivo study, the administration of EG induced renal calcium crystallization (Fig. 4) and significant oxidative stress as evaluated by SOD, MDA, and 8-OHdG expression (Fig. 5). However, CsA significantly inhibited renal calcium crystallization and oxidative stress. Additionally, in the EG + CsA groups, mitochondria showed a regular internal structure with a continuous double membrane similar to that of mitochondria in the control group (Fig. 6). These results indicated that CsA prevented mitochondrial collapse by blocking mPTP opening, which was induced by EG. The expression of OPN was evenly and densely distributed throughout renal tubular cells of whole kidneys in the EG group.

K. Niimi et al. / Free Radical Biology & Medicine 52 (2012) 1207–1217

However, in the control group and in the EG + CsA groups, the expression of OPN was barely detectable or localized to limited renal tubular cells (Fig. 4). Not only was OPN identified as a major component of stone matrix protein, but its expression was also remarkably increased in the renal tubular cells of stone-forming rats [13,35]. Some studies suggested that OPN was an inhibitor of abnormal calcification in rat kidneys [36,37]. However, other studies suggested that OPN plays a role in stimulating the deposition and adhesion of crystals to cells in the early stages of crystallization [38] and showed that calcium oxalate monohydrate crystal coating with OPN correlated with increased adhesion tendency [39]. Furthermore, we have reported that kidney crystal deposition was decreased in OPN-deficient mice compared to crystal deposition in wild-type mice [40,41]. The therapeutic benefits of mPTP inhibitors such as CsA have recently been described. For example, mPTP inhibition reduced cholestatic liver damage in a mouse model [42] and mitochondrial injury in the ischemia-reperfused liver after transplantation [43]. CsA decreased myocardial infarction during percutaneous coronary artery intervention [44] and might therefore serve as a drug for treating degenerative nerve diseases [45–47]. These diseases are associated with cell injury induced by mitochondrial collapse. High dose and long-term use of CsA are known to cause chronic nephrotoxicity [48]. A CsA dose of 25 mg/kg/day for 21 days [49] and 100 mg/kg/day for 7 days [50] by gastric tube induced chronic nephrotoxicity in the rat. In this study, the CsA doses used were between 2.5 and 10 mg/kg/day for 14 days, which was a low dose and short treatment period compared to the dosages in those reports,

Change of Phosphatidylserine distribution

(9)

NADPH oxidase NADPH e NADP+H+

(2)

O2

and there were no rats showing signs of chronic nephrotoxicity. The low dose of CsA was therefore essential to obtain good results and avoid chronic nephrotoxicity. Low-dose CsA was very important in this study. A model of the proposed pathway of renal crystallization based on the present result is shown in Fig. 7. COM crystals attach to renal tubular cells and NADPH oxidase generates O2•− [31], which activates cyclophilin D and leads to mPTP opening accompanied by mitochondria collapse. Cyclosporine A blocks mPTP opening by inactivating cyclophilin D [23]. O2•− released from mitochondria decreases SOD and increases 8-OHdG, 4-HNE, and MDA. Cytochrome c, which is also released from mitochondria, activates caspase 3. These events trigger the onset of apoptosis and cell injury and change phosphatidylserine distribution in renal tubular cell membranes, which causes an increase in crystal attachment and renal calcium crystallization [8–10]. CsA can reduce oxidative stress and prevent the apoptotic signal by blocking mPTP opening, thus preventing subsequent renal calcium crystallization. In addition to oxidative stress and renal tubular cell injury, mitochondria might participate in renal calcium crystallization via another mechanism. Mitochondria store a considerable amount of calcium ions, which are discharged from mitochondria into the cytosol as a result of mitochondrial collapse. This movement of calcium ions is important because crystals are primarily composed of calcium, and elevation of cytosolic calcium is therefore significant. From the above, we suggest that inhibition of oxidative stress and apoptosis, caused by mitochondrial collapse, controls crystal cell interaction and renal calcium crystallization.

Renal crystallization (Crystal attachment)

COM crystals (1)

(8)

(7)

(6) Cytocrome c

O2

Apoptosis (Cell injury)

Caspase 3

(3) Activation of Cyclophilin D

1215

(5-c)

(5)

Opening of MPTP

Mitochondrial collapse

4 HNE MDA

O2 8OHdG

Mitochondria

(4)

H2O

Cyclosporine A

OH

(5-b)

(5-a) H2O2

Cu-Zn SOD

Nucleus

Renal tubular cell

Fig. 7. A proposed scheme of the pathway of renal calcium crystallization. Boxes with a solid-line border indicate the findings in this study (1, 3, 5-a, 5-b, 5-c, 6, 9). Boxes with a dashed-line border indicate the findings from reference citations (2, 5, 7, 8) [8–10,23,30,34]. The pathway depicted is proposed from the results of this study. (1) COM crystals attach to renal tubular cells, (2) NADPH oxidase [32] generates superoxide (O2•−), which (3) activates cyclophilin D and leads to mPTP opening accompanied by mitochondria collapse. (4) Cyclosporine A blocks mPTP opening by inactivating cyclophilin D [23]. (5) O2•− released from mitochondria [30] (5-a) decreases SOD and increases (5-b) 8-OHdG and (5-c) 4-HNE and MDA. (6) Cytochrome c, also released from mitochondria, activates caspase 3. (7) These events activate apoptosis and cell injury [34] and (8) alter phosphatidylserine distribution in renal tubular cell membranes, which (9) increases crystal attachment and renal calcium crystallization [8–10].

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In conclusion, this study provides compelling evidence for the involvement of mPTP opening in the initial process of renal calcium crystallization. Blocking mPTP opening by CsA prevents renal calcium crystallization caused by mitochondrial collapse, oxidative stress, and activation of the apoptosis pathway. Targeting mPTP opening by pharmacological and genetic approaches may offer a new therapeutic strategy for renal calcium crystallization.

[23]

[24]

[25]

Acknowledgments This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Nos. 18209050, 20591887, and 22791484); the 24th General Assembly of the Japanese Association of Medical Sciences; and The Hori Information Science Promotion Foundation.

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