Manganese-Induced Nephrotoxicity Is Mediated through Oxidative Stress and Mitochondrial Impairment

Manganese (Mn) is an essential element that is incorporated in various metabolic pathways and enzyme structures. On the other hand, a range of adverse effects has been described in association with Mn overexposure. Mn is a well-known neurotoxic agent in mammals. Renal injury is another adverse effect associated with Mn intoxication. No precise mechanism for Mn nephrotoxicity has been identified so far. The current study was designed to evaluate the potential mechanisms of Mn-induced renal injury. Rats were treated with Mn (20 and 40 mg/mL, respectively, in drinking water) for 30 consecutive days. Markers of oxidative stress, as well as several mitochondrial indices, were assessed in the kidney tissue. Renal injury was evident in Mn-treated animals, as judged by a significant increase in serum BUN and creatinine. Moreover, urinalysis revealed a significant increase in urine glucose, phosphate, and protein in Mn-treated rats. Kidney histopathological alterations, including tubular atrophy, interstitial inflammation, and necrosis, were also detected in Mn-treated animals. Biomarkers of oxidative stress, including an increment in reactive oxygen species (ROS), lipid peroxidation, and oxidized glutathione (GSSG), were detected in Mn-treated groups. On the other hand, kidney glutathione (GSH) stores and total antioxidant capacity were depleted in Mn groups. Mn exposure was associated with significant mitochondrial depolarization, decreased mitochondrial dehydrogenases activity, mitochondrial permeabilization, and depletion of adenosine tri-phosphate (ATP) content. These data highlight oxidative stress and mitochondrial impairment as potential mechanisms involved in Mn-induced renal injury.

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- . Animals were allotted to three groups (n = 8 in each group), and treated as follows: (i) Control (vehicle-treated); (ii) MnCl 2 (20 mg/mL in drinking water); and (iii) MnCl 2 (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.

Sample collection
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 and mitochondria glutathione content
Renal glutathione levels (oxidized and reduced; GSSG and GSH) were measured by the HPLC method (23). The HPLC plays a central role in the adverse effects of this metal on the nervous system (9)(10)(11)(12). It has been reported that Mn accumulates in cellular mitochondria through calcium (Ca 2+ ) channels (2). Hence, cellular mitochondria are critical targets for Mn cytotoxicity. Mitochondrial depolarization, mitochondria swelling, increased mitochondria-mediated reactive oxygen species (ROS) formation, and mitochondriamediated cell death have been reported in different experimental models that investigated Mn neurotoxicity (9)(10)(11)(12)(13).
Renal tissue contains numerous mitochondria, the proper functioning of which guarantee appropriate energy (ATP) level required for the reabsorption process of chemicals (14)(15)(16). The mechanism of nephrotoxicity induced by several xenobiotics relies on mitochondrial impairment and mitochondria-mediated cell death (17). Oxidative stress and mitochondrial injury are two mechanistically related events (18). Hence, mitochondrial impairment could deteriorate oxidative stress and vice versa.
As already mentioned, there is no precise mechanism for Mn-induced renal injury. The current study was designed to evaluate the role of oxidative stress and mitochondrial impairment in the pathogenesis of Mn nephrotoxicity. Rats were exposed to Mn for 30 consecutive days. Several biomarkers in serum and urine, as well as histopathological alterations and oxidative stress markers in renal tissue, were evaluated. Moreover, kidney tissue mitochondria were isolated and assessed.

Kidney mitochondria isolation
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 KH 2 PO 4 , 2 mM MgCl 2 , 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. system consisted of an NH 2 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: NaHCO 3 (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).

Ferric reducing antioxidant power
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 (Fe 2+ )-4, 6-tripyridyl-s-triazine (TPTZ) complex from the colorless oxidized ferric form (Fe 3+ ) 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 (FeCl 2 , 20 mmol/L). Tissue samples (200 mg) were homogenized in 5 mL of 250 mM Tris-HCl buffer

Mitochondrial depolarization
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 permeabilization and swelling
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).

Statistical methods
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.

Results
Animal weight gain was significantly lower in the Mn-treated group (40 mg/mL) in comparison with control rats ( Figure 1A). The kidney WI was also significantly lower in Mn 40 mg/mL group ( Figure 1A). Serum and kidney tissue Mn levels were also significantly higher in Mn-treated animals ( Figure 1B).
Significant deterioration in serum biochemical measurements indicates renal injury in Mn-treated rats ( Table 1). Signs of hypophosphatemia were evident in the Mn group (Table 1). On the other hand, serum BUN and creatinine levels were significantly higher in Mn-exposed animals (20 and 40 mg/mL) ( Table 1). Significant elevation in urine protein, alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), and glucose level was also detected in Mn-treated rats ( Table 2).    Data are given as mean ± SD (n = 8).
Significant ROS formation, lipid peroxidation, and protein carbonylation were detected in Mn groups ( Figure 2). Moreover, kidney tissue GSH was depleted, and the GSSG level was significantly increased in Mn-exposed animals ( Figure 2). Tissue antioxidant capacity was also dose-dependently decreased in the kidneys of Mn-treated rats (Figure 2).
Several mitochondrial indices were assessed in the kidney tissue of Mn-treated rats (Figure 3). It was found that Mn exposure significantly decreased mitochondrial dehydrogenases activity and ATP levels in a dose-dependent manner ( Figure 3). Moreover, a significant increment of mitochondrial depolarization and swelling was detected in the kidney mitochondria of Mn-treated animals ( Figure 3). Lipid peroxidation was also dose-dependently increased in the kidney mitochondria isolated from Mn-exposed animals ( Figure 3).
Significant interstitial inflammation and tubular atrophy were evident in the kidneys of Mn 20 and 40 mg/mL groups ( Figure 4 and Table 3). Moreover, Mn 40 mg/mL caused renal tissue necrosis ( Figure 4 and Table 3). Data are presented as mean ± SD (n = 8). *** Significantly different from the control group (P < 0.001).

Control
Min 20 mg/ml Min 40 mg/ml Figure 4. Kidney tissue histopathological alterations in manganese-exposed animals. Hematoxylin and eosin staining. The grades of histopathological changes are given in Table 3. Table 3. Renal tissue histopathological alterations in manganese-exposed rats.

Discussion
Mn is a trace element that plays a fundamental role in several metabolic pathways and enzyme structures (2). However, overexposure to this metal is associated with a wide range of adverse effects, including renal injury (6)(7)(8)38). Acute tubular necrosis, proteinuria, oliguria, and significant elevation in serum creatinine levels have been reported in human cases of Mn-induced nephrotoxicity (6,(38)(39)(40). No precise mechanism for Mn-induced nephrotoxicity has been identified so far. In the current investigation, it was found that Mn caused significant oxidative stress as well as mitochondrial impairment in the kidney tissue. The results might help in the development of therapeutic options against renal failure and serum electrolyte imbalance observed in Mn-intoxicated patients (6,(38)(39)(40)(41).
Neurotoxicity is a well-described adverse effect of Mn (9,13,(42)(43)(44)(45). Oxidative stress and its consequences, such as disruption of biomembrane lipids and protein carbonylation, seem to play a fundamental role in Mn-induced toxicity in different organs such as the brain (9,13,(42)(43)(44)(45). Severe elevation in brain tissue ROS level and lipid peroxidation has been documented in Mn-induced neurotoxicity (9,13,(42)(43)(44)(45). Moreover, it has been found that brain tissue antioxidant systems are hampered upon Mn overexposure (9,13,(42)(43)(44)(45). The mechanism(s) of nephrotoxicity induced by Mn is less understood. Previous studies mentioned the occurrence of oxidative stress in the renal tissue of Mn-exposed animals (7). Mn-induced oxidative stress could affect several cellular targets, including biomembrane lipids, proteins, as well as deoxyribonucleic acid (7). In the current study, significant ROS formation, protein carbonylation, lipid peroxidation, and depletion of kidney tissue antioxidant capacity was evident in Mn-exposed rats. These results are consistent with previous investigations indicating Mn-induced oxidative stress in the kidney (7). Moreover, we found that kidney mitochondria could also be affected by Mn overexposure.
Cellular mitochondria are critical targets affected by Mn. (13). It has been found that Mn is accumulated in the mitochondrial matrix through Ca 2+ channels (13). Induction of mitochondrial permeabilization, enhancement of mitochondria-facilitated ROS formation, a decrease of cellular ATP levels, and mitochondria-mediated cell death and apoptosis are associated with Mn-induced mitochondrial impairment (13).
Mitochondria play a fundamental role in kidney tissue (14)(15)(16). The reabsorption of chemicals (e.g., amino acids, glucose, and minerals) from nephrons to the bloodstream is an energy-dependent activity (14)(15)(16). Kidney tissue contains numerous mitochondria, the proper functioning of which guarantee enough ATP required for the reabsorption process of chemicals (14)(15)(16). Hence, Mn-induced mitochondrial impairment leads to an energy crisis and defect in the reabsorption of many chemicals in the renal tubules.
Consequently, serum electrolyte disturbances could occur. Cellular mitochondria are also important sites of ROS production (18). It has been repeatedly reported that xenobiotics-induced mitochondrial impairment could facilitate mitochondria-mediated ROS formation (18). Based on the data obtained from the current study, we might be able to speculate that Mn-induced mitotoxicity could serve as a major cause of oxidative stress in the renal tissue.

Conclusion
Collectively, our results indicate the fundamental role of oxidative stress and mitochondrial impairment in the pathogenesis of Mn-induced renal injury. Therefore, targeting cellular mitochondria might serve as a therapeutic point against Mn-induced nephrotoxicity.