3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), iodoacetic acid, potassium hydroxide, bovine serum albumin (BSA), methanol high performance liquid chromatography (HPLC) grade, 3-(N-morpholino) propane sulfonic acid (MOPS), 4,2Hydroxyethyl,1-piperazineethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), thiobarbituric acid (TBA), glacial acetic acid, glutathione (GSH), malondialdehyde (MDA), 2′,7′ Dichlorofluorescein diacetate (DCFH-DA), acetonitrile HPLC grade, ethylene glycol-bis (2-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), sucrose, dithiothreitol (DTT), sodium chloride, and Rhodamine 123 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Manganese chloride (MnCl₂), trichloroacetic acid (TCA), and hydroxymethyl aminomethane hydrochloride (Tris-HCl) were purchased from Merck (Darmstadt, Germany).

Mature male Sprague–Dawley rats (200–250 g, live weight; n = 24) were obtained from Shiraz University of Medicine, Shiraz, Iran. Rats were maintained under standard conditions (22–24°C; 12:12 h, photo schedule; appropriate ventilation; and 40 ± 2% relative humidity). Animals had free access to tap water and a commercial rodents chow diet (RoyanFeed^®, Isfahan, Iran). All procedures on experimental animals were performed in compliance with the ethical guidelines approved by the Shiraz University of Medical Sciences ethics committee (#95-01-36-11290). Animals were allotted to three groups (n = 8 in each group), and treated as follows: (i) Control (vehicle-treated); (ii) MnCl₂ (20 mg/mL in drinking water); and (iii) MnCl₂ (40 mg/mL in drinking water). Animals were treated for 42 consecutive days. On day 43, rats were anesthetized, and serum and kidney tissue samples were collected.

Animals were anesthetized (Thiopental 80 mg/kg, i.p). Blood was collected from the abdominal aorta and transferred to standard tubes (VACUSERA^®, Serum gel, and clot activator tubes) for serum preparation. The kidney tissue was washed in ice-cooled (4°C) normal saline and used for further assessments. Kidney weight index (WI) was determined as WI = [wet weight of organ (g)/body weight (g)] × 100.

Renal tissue ROS levels were measured using dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe (19). Briefly, 10 μL of DCFH-DA (10 μM final concentration) was added to 990 μL of tissue homogenate (10% w: v in KCl buffer). Samples were incubated for 15 min at 37°C in the dark. Finally, the DCF fluorescence intensity was assessed using a fluorimeter (FLUOstar Omega^®, BMG LABTECH, Germany; λ_excit = 485 nm and λ_em = 525 nm) (19).

Thiobarbituric acid reactive substances (TBARS) in renal tissue were measured as an index of lipid peroxidation in the renal tissue (12, 20). Briefly, renal tissue (500 mg) was homogenized in ice-cooled KCl buffer (1.15% w: v; 4°C). Then, 500 µL of tissue homogenate was added to a reaction mixture consisting of 1 mL TBA (0.375%, w: v), 1 mL trichloroacetic acid (20% w: v), and meta-phosphoric acid (3 mL f 1% w: v solution, pH = 2) (21). The mixture was incubated in a water bath (100°C) for 45 min (22). Then, 4 mL of n-butanol was added and vortexed (1 min). Finally, samples were centrifuged (17,000 × g for 10 min) and the absorbance of the developed color in upper phase (n-butanol) was measured at λ = 532 nm (EPOCH^® plate reader, Highland Park, USA) (21).

Renal glutathione levels (oxidized and reduced; GSSG and GSH) were measured by the HPLC method (23). The HPLC system consisted of an NH₂ column as the stationary phase (25 cm, Bischoff chromatography, Leonberg, Germany) (24). The mobile phases consisted of buffer A (Water: Methanol; 1: 4 v: v) and buffer B (Acetate buffer: Buffer A; 1: 4 v: v), and a gradient method with a steady increase of buffer B to 95% in 25 min (24). The flow rate of the mentioned mobile phase was 1 mL/min, and the UV detector (UV) detector was set at λ = 254 nm. Tissue samples were homogenized in Tris-HCl buffer (250 mM; pH = 7.4; 4°C), and 500 µL of TCA (50% w: v) was added. Isolated mitochondria (1 mL, 1 mg protein/mL) were also treated with 100 µL of TCA 50% w: v. Samples were mixed well, incubated on ice (10 min), and centrifuged (17,000 g, 30 min, 4°C). Afterward, 1 mL of the supernatant was collected in 5 mL tubes and 300 µL of the NaOH: NaHCO₃ (2 M: 2 M) solution was added. Then, 100 µL of iodoacetic acid (1.5% w: v in deionized water) was added, and samples were incubated for 1 h (4°C, in the dark). Afterward, 2, 4-dinitrofluorobenzene (DNFB, 500 µL of 1.5% w: v in absolute ethanol) was added and mixed well. Samples were incubated in the dark (25°C, 24 h). Finally, samples were centrifuged (17,000 g, 30 min) and injected (25 µL) into the described HPLC system (23).

The oxidative damage of kidney tissue proteins was assessed based on the reaction with dinitrophenyl hydrazine (DNPH) (25). Briefly, 1 mL of the tissue homogenate (10% w: v) was treated with 100 µL of the triton X-100 (0.1% w: v) and centrifuged (10 min, 700 g, 4°C). Then, 500 µL aliquots of the resulting supernatant were treated with 300 µL of DNPH (10 mM in HCl). Samples were then incubated for 1 h (25°C, vortexing every 10 min). Afterward, 100 µL of trichloroacetic acid (20% w: v) was added and centrifuged (12,000 g, 5 min) (26). The pellet was washed three times with ethanol: ethyl acetate (1 mL of 1: 1 v: v solution). Finally, the precipitate was re-dissolved in guanidine hydrochloride solution (600 µL of 6 M solution), and the absorbance at λ = 370 nm was measured (EPOCH^® plate reader, Highland Park, USA) (25).

The ferric reducing antioxidant power (FRAP) of kidney tissue was measured (27). The FRAP assay measures change in the absorbance at λ = 593 nm due to the formation of a blue-colored ferrous (Fe²⁺)-4, 6-tripyridyl-s-triazine (TPTZ) complex from the colorless oxidized ferric form (Fe³⁺) by the action of tissue electron-donating antioxidants. The working FRAP solution was freshly prepared by mixing 25 mL of acetate buffer (300 mmol/L; pH = 3.6) with 2.5 mL of TPTZ ( 10 mmol/L in 40 mmol/L HCl) and 2.5 mL of ferric chloride (FeCl₂, 20 mmol/L). Tissue samples (200 mg) were homogenized in 5 mL of 250 mM Tris-HCl buffer (pH = 7.4; 4°C). Afterward, 50 µL of tissue homogenate was added to 900 µL of the FRAP reagent and incubated in the dark (37°C, 5 min). The intensity of the resultant blue color was measured at λ = 593 nm using an EPOCH plate reader (BioTek^® Instruments, Highland Park, US) (27).

For histopathological evaluations, renal specimens were fixed in a buffered formalin solution prepared from NaH₂PO₄ (0.4% w: v), Na₂HPO₄ (0.64% w: v), and formaldehyde (10%) in double-distilled water (pH = 7.4). Paraffin-embedded tissue specimens were cut (5 µm) using a microtome and stained with hematoxylin and eosin. Kidney histopathological changes were evaluated using a light microscope (Olympus BX41; Olympus Optical Co. Ltd, Japan).

Serum and kidney Mn levels were measured using an inductively coupled plasma mass spectrometry (ICP-MS) method (28). Briefly, serum (500 µL) and kidney tissue samples (500 µL of 10% w: v tissue homogenate) were treated with 100 µL of nitric acid (HNO₃; 2.5% w: v) and centrifuged (17,000 g, 30 min). The supernatants were collected and used for Mn determination (28).

Rats’ kidneys were washed (NaCl 0.9% w: v, 4°C) and minced in the ice-cold isolation buffer containing 0.5 mM EGTA, 2 mM HEPES, 220 mM sucrose, 70 mM mannitol, and BSA (0.1% w: v) (pH = 7.4). Minced tissue was transported into fresh isolation buffer (5 mL buffer: 1 g tissue) and homogenized. Kidney mitochondria were isolated based on the differential centrifugation method (29). For this purpose, unbroken cells and nuclei were pelleted at the first round of centrifugation (1000 g for 10 min at 4°C). Afterward, the supernatant was centrifuged at 10,000 g (10 min at 4°C) to pellet the mitochondria fraction (brown-colored). The second centrifugation step was repeated at least thrice using a fresh buffer medium. Finally, mitochondrial pellets were resuspended in a buffer (5 mL buffer/g tissue) containing 70 mM mannitol, 220 mM sucrose, and 2 mM HEPES (pH = 7.4). The mitochondria fractions used to measure mitochondrial permeabilization and mitochondrial depolarization were suspended in mitochondria permeabilization buffer (65 mM KCl, 10 mM HEPES, 125 mM Sucrose, pH = 7.2) and depolarization assay buffer (220 mM Sucrose, 10 mM KCl, 68 mM Mannitol, 5 mM KH₂PO₄, 2 mM MgCl₂, 50 μM EGTA, and 10 mM HEPES, pH = 7.2) (29). Sample protein concentrations were determined based on the Bradford method to standardize the obtained data.

A colorimetric technique based on the production of purple formazan crystals from the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide was used for the estimation of mitochondrial dehydrogenases activity (30). Briefly, a mitochondrial suspension (0.5 mg protein/mL) was incubated with 40 µL of MTT (0.4% w: v) and incubated in the dark (37°C, 30 min). Samples were centrifuged (15,000 g, 15 min), and the pellet was dissolved in dimethyl sulfoxide (DMSO; 1000 µL). The optical density (OD) at λ = 570 nm was measured with an EPOCH^® plate reader (Highland Park, USA) (30).

Based on a previously reported protocol, mitochondrial ATP level was assessed by HPLC (31, 32). Briefly, isolated mitochondria (1 mg protein/mL) were mixed with 100 µL ice-cooled meta-phosphoric acid solution (50% w: v, 4°C) and centrifuged (30 min, 17,000 g, 4°C). Afterward, the supernatant (100 µL) was treated with 15 µL of ice-cooled potassium hydroxide solution (KOH, 1 M). Samples were centrifuged (30 min, 17,000 g, 4°C) again, and 25 µL of the supernatant was injected into an HPLC system composed of an LC-18 column (µ-Bondapak, 25 cm). The mobile phase was composed of potassium hydrogen phosphate mono-basic (100 mM KH₂PO₄, pH = 7 adjusted with KOH), acetonitrile (2.5% v: v), and tetrabutylammonium hydroxide (1 mM). The flow rate was 1 mL/min, and the UV detector was set at λ = 254 nm (31).

Mitochondrial uptake of the cationic dye rhodamine 123 was applied for the evaluation of mitochondrial depolarization (33, 34). Rhodamine 123 accumulates in the mitochondrial matrix by facilitated diffusion. When the mitochondrion is depolarized, there is no facilitated diffusion, and the amount of rhodamine 123 in the supernatant will be increased (35). In the current investigation, the mitochondrial fractions (0.5 mg protein/mL; in the depolarization assay buffer) were incubated with 10 µM of rhodamine 123 (30 min, 37°C, in the dark). Afterward, samples were centrifuged (15,000 g, 10 min, 4°C) and the fluorescence intensity of the supernatant was monitored with a fluorimeter (FLUOstar Omega^®; BMG, Germany; λ_excit = 485 nm and λ_em = 525 nm) (33, 36).

Mitochondrial swelling was estimated by analyzing the changes in optical density at λ = 540 nm (34, 37). Briefly, isolated mitochondria (0.5 mg protein/ml) were suspended in the mitochondria permeabilization buffer (65 mM KCl, 125 mM Sucrose, 10 mM HEPES, pH = 7.2), and the absorbance was monitored (30°C, during 30 min of incubation) using an EPOCH^® plate reader (Highland Park, USA). An increase in mitochondrial swelling is associated with a decrease in absorbance. The results are reported as maximal mitochondrial swelling amplitude (ΔOD 540 nm) (37).

TBARS were measured in isolated kidney mitochondria. Previous studies mentioned that sucrose interferes with the lipid peroxidation test in isolated mitochondria preparations (37). Therefore, mitochondria preparations were washed once (to remove sucrose) in ice-cooled MOPS-KCl buffer (100 mM KCl, 50 mM MOPS, 4°C, pH = 7.4). For this purpose, isolated kidney mitochondria were suspended in 5 mL of MOPS-KCl buffer and centrifuged (15,000 g, 4°C, 20 min). The pellet was re-suspended in MOPS-KCl buffer and used for TBARs assay. The mitochondrial suspension (1 mg protein/mL) was mixed with 1 mL of a solution containing trichloroacetic acid (15% w: v), HCl (0.24 N), TBA (0.375% w: v), and Trolox (500 µM). Samples were heated for 15 min at 100°C (37). Then n-butanol (1 mL) was added and vortexed (5 min). Samples were centrifuged (15,000 g, 10 min), and the absorbance of the n-butanol phase (upper phase) was measured (EPOCH^® plate reader, Highland Park, USA, λ = 532 nm) (37).

Data are represented as mean ± SD. Data analysis was accomplished by the one-way analysis of variance (ANOVA) and the Tukey’s multiple comparison test as the post hoc test. A P < 0.05 was considered as a statistically significant difference.