Nephrology ••23(2017)

Nephrology (2018)••–••
351–361

Original Article

Mechanism of valproic acid-induced Fanconi syndrome involves

mitochondrial dysfunction and oxidative stress in rat kidney
REZA HEIDARI,1 FAEZEH JAFARI,2 FOROUZAN KHODAEI,2 BABAK SHIRAZI YEGANEH3 and HOSSEIN NIKNAHAD1,2
1
Pharmaceutical Sciences Research Center, 2Department of Pharmacology and Toxicology, School of Pharmacy, and 3Department of Pathology, School of Medicine,
Shiraz University of Medical Sciences, Shiraz, Iran

KEY WORDS: ABSTRACT:

carnitine, energy metabolism, Fanconi syndrome,
mitochondria, oxidative stress, renal injury.
Correspondence:
Professor Hossein Niknahad, Pharm. D and
Toxicology Phd, Pharmaceutical Sciences
Research Center, Shiraz University of Medical
Sciences, P. O. Box 1583; 71345, Roknabad,
Karafarin St., Shiraz, Fars, Iran.
Email: niknahadh@sums.ac.ir
Accepted for publication 30 January 2017.
Accepted manuscript online 31 January 2017.
doi: 10.1111/nep.13012
SUMMARY AT A GLANCE
Valproic acid is a broad-spectrum antiepilep-
tic drug with nephrotoxic properties. Using
an animal model, Heidari et al. shed light on
the role of mitochondrial function and oxida-
tive stress in its pathogenesis.
Aim: Drug-induced kidney proximal tubular injury and renal failure (Fanconi
syndrome; FS) is a clinical complication. Valproic acid (VPA) is among the FS-
inducing drugs. The current investigation was designed to evaluate the role
of mitochondrial dysfunction and oxidative stress in VPA-induced renal
injury.
Methods: Animals received VPA (250 and 500 mg/kg, i.p., 15 consecutive days).
Serum biomarkers of kidney injury and markers of oxidative stress were
assessed. Moreover, kidney mitochondria were isolated and mitochondrial
indices, including succinate dehydrogenase activity (SDA), mitochondrial
depolarization, mitochondrial permeability transition pore (MPP), reactive
oxygen species (ROS), lipid peroxidation (LPO), mitochondrial glutathione,
and ATP were determined.
Results: Valproic acid-treated animals developed biochemical evidence of FS
as judged by elevated serum gamma-glutamyl transferase (γ-GT), alkaline
phosphatase (ALP), creatinine (Cr), and blood urea nitrogen (BUN) along with
hypokalaemia, hypophosphataemia, and a decrease in serum uric acid. VPA
caused an increase in kidney ROS and LPO. Renal GSH reservoirs were
depleted and tissue antioxidant capacity decreased in VPA-treated animals.
Renal tubular interstitial nephritis, tissue necrosis, and atrophy were also
evident in VPA-treated rats. Mitochondrial parameters including SDA, MMP,
GSH, ATP and MPP were decreased and mitochondrial ROS and LPO were
increased with VPA treatment. It was found that carnitine (100 mg/kg, i.p.)
mitigated VPA adverse effects towards the kidney.
Conclusions: These data suggest that mitochondrial dysfunction and oxidative
stress contributed to the VPA-induced FS. On the other hand, carnitine could
be considered a potentially safe and effective therapeutic option in
attenuating VPA-induced renal injury.

Valproic acid (2-propyl-pentanoic acid, VPA) is widely adminis-
tered in epilepsy and psychiatric disorders. Although VPA has a
good pharmacological efficacy and a relatively favourable
safety profile, several adverse drug reactions have been
reported in relation to VPA therapy. Hepatotoxicity,
hyperammonaemic encephalopathy, hypersensitivity reac-
tions, weight gain, pancreatitis, haematological abnormalities,
gastrointestinal disturbances, neurological toxicity, as well as
renal injury, have been reported after VPA administration.1,2
Several investigations indicated the injurious effect of VPA on
the kidney in human and animal models.3–9 There is no clear
mechanism for VPA-induced renal injury.
Fanconi syndrome (FS) is characterized by a generalized
transport defect in the renal proximal tubules leading to the
impaired renal tubular transport of ions and electrolytes.10
The occurrence of FS results in patients’ dehydration, electro-
lytes abnormalities, and altered mental status.1,10 A number
of inherited metabolic defects, as well as toxins and drugs, can
cause FS.10 Heavy metals, antibiotics, anticonvulsants, and
ifosfamide are among FS-inducing agents.10 FS is also a well-
described complication of VPA therapy.1,8,10 VPA-induced
renal injury can manifest as proximal tubular dysfunction.10
The pathophysiological characteristics of VPA-induced FS are
not understood well.

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Hepatotoxicity is a well-recognized adverse effect of VPA.1,2
VPA hepatotoxicity is believed to be mediated by its effect on
hepatocytes mitochondria.11 VPA-induced inhibition of mito-
chondrial β-oxidation of fatty acids, impairment of gluconeo-
genesis, interference with urea synthesis, and inhibition of
oxidative phosphorylation have been reported in liver prepara-
tions.11–13 The involvement of mitochondria in the pathophys-
iology of VPA-induced FS has not been evaluated so far. On the
hand, supplementation with carnitine offers beneficial effects
in patients with VPA-induced liver injury.14 Hence, we investi-
gated whether carnitine could improve VPA-induced FS in this
study.
Understanding the mechanism(s) of VPA-induced FS can be
useful for developing new preventive/therapeutic strategies
against this complication. The current study was designed to
evaluate the effect of VPA on rat kidney. Different serum and
tissue biomarkers, along with the effect of VPA on kidney mito-
chondria have been evaluated here. Moreover, the potential
protective effect of carnitine on the VPA-induced renal injury
was investigated in this study.
MATERIALS AND METHODS
Chemicals
20 ,70 -Dichlorofluorescein diacetate (DCFH-DA), Trichloroacetic
acid (TCA), Valproic acid (VPA), Glutathione (GSH),
Malondialdehyde (MDA), 3-[4,5dimethylthiazol-2-yl]-2,5-di-
phenyltetrazolium bromide (MTT), Sucrose, D-Mannitol,
3-(N-morpholino) propane sulfonic acid (MOPS), Fatty acid-
free bovine serum albumin (BSA) fraction V, Rhodamine123
(Rh 123), Coomassie brilliant blue, 2, 4, 6-tripyridyl-s-triazine
(TPTZ), Ferric chloride hexahydrate (FeCl3.6H2O), Dithiothrei-
tol (DTT), 6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid (Trolox), and Thiobarbituric acid (TBA) were
purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA).
Kits for evaluating serum biomarkers of renal injury were
obtained from Pars Azmun (Tehran, Iran). Ethylenediamine-
tetraacetic acid (EDTA), 5, 5-bis-dithio-nitro benzoic acid
(DTNB), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), meta-Phosphoric acid, n-Butanol, and 2-Amino-2-
hydroxymethyl-propane-1, 3-diol-hydrochloride (Tris–HCl),
were obtained from Merck (Darmstadt, Germany). All salts
used for making buffer solutions were of analytical grade and
obtained from Merck (Darmstadt, Germany).
Animals
Male Sprague–Dawley rats (n = 32) weighing 200–250 g were
obtained from the Laboratory Animal Breeding Center, Shiraz
University of Medical Sciences, Shiraz, Iran. Animals were
housed in an environmental temperature of 23 ± 1°C with a
40% of relative humidity. Rats had free access to tap water
and a standard pellet chow during experiments. All procedures
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involving animal use were in accordance with the guidance for
care and use of experimental animals and were approved by a
local ethic committee in Shiraz University of Medical Sciences,
Shiraz, Iran (94–01–36-10650).
Experimental setup
Animals were randomly allotted into four groups (n = 8 in each
group). Rats were treated as follows: (i) Control (Vehicle-
treated group), (ii) VPA (250 mg/kg per day, i.p); (iii) VPA
(500 mg/kg per day, i.p); (iv) VPA (500 mg/kg per day, i.
p) + Carnitine (100 mg/kg per day, i.p). Normal saline was used
as valproic acid and carnitine vehicle (2.5 mL/kg). Carnitine
was administered 2 h after VPA. In the current investigation,
carnitine (100 mg/kg, i.p) cause no kidney injury when it was
administered alone. It has been previously reported that a dose
of 500 mg/kg per day of valproic acid for 15 consecutive days
caused marked renal injury in rats.15 At the end of experiments
(At day 16th, 24 h after the final dose of VPA), animals were
anesthetized (ketamine/xylazine; 100/10 mg/kg, i.p) and their
blood and kidney were collected.
Serum biochemistry
Blood was collected from the abdominal aorta, transferred to
standard tubes (Improvacuter; gel and clot activator-coated
tubes; Guangzhou, China) and centrifuged (3000 g, 10 min,
4°C) to prepare serum. A Mindray BS-200 auto analyzer and
standard kits were employed to assess serum gamma-glutamyl
transpeptidase (γ-GT), alkaline phosphatase (ALP), Creatinine
(Cr), glucose, phosphate, calcium, uric acid and blood urea
nitrogen (BUN).16 Serum sodium and potassium level were
assessed by a flame photometer (Clinical Flame Photometer,
Sherwood Scientific, UK).
Kidney histopathology
For histopathological assessments, samples of kidney tissue
were fixed in a buffered formalin solution (0.4% sodium phos-
phate monobasic, NaH2PO4, 0.64% sodium phosphate dibasic,
Na2HPO4, and 10% formaldehyde in distilled water; pH = 7.4).
Finally, paraffin-embedded sections (5 μm) of tissue were
stained with haematoxylin and eosin (H&E) and Masson
trichrome (for renal interstitial fibrosis).17 Renal histopatholog-
ical changes were scored as previously described based on a
model of toxic nephropathy.18 Samples were analyzed by a
pathologist in a blind fashion.
Kidney tissue reactive oxygen species (ROS)
formation
Kidney samples (200 mg) were homogenized in ice-cooled
Tris–HCl buffer (40 mM, pH = 7.4) (1:10 w/v). Then, 100 μL
of tissue homogenate was mixed with 1 mL of Tris–HCl buffer
(40 mM, pH = 7.4) and 5 μL of 20 , 70 -dichlorofluorescein
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diacetate (Final concentration 10 μM). The mixture was incu-
bated in dark for 30 min at 37°C (Gyromax incubator shaker).
Finally, the fluorescence intensity of the samples was assessed
using an FLUOstar Omega multifunctional microplate reader
(λ excitation = 485 nm and λ emission = 525 nm).16,19
Tissue lipid peroxidation
Thiobarbituric acid reactive substances (TBARs) were assessed
in kidney tissue as an index of lipid peroxidation. Briefly,
1 mL of kidney tissue homogenate (10% w/v in KCl, 1.15%)
was added to a reaction mixture consisted of thiobarbituric acid
(0.375%, w/v), trichloroacetic acid (15%; w/v), and hydrochlo-
ric acid (0.2 N) (pH = 2). Samples were mixed well and heated
in boiling water (100°C, 45 min).20 Finally, 2 mL of n-butanol
was added and vigorously mixed. Samples were centrifuged
(3000 g for 5 min) and the absorbance of developed color in
n-butanol phase was measured at 532 nm using an Ultrospec
2000®UV spectrophotometer (Uppsala, Sweden).21
Kidney glutathione content
Tissue samples (200 mg) were homogenized in 8 mL of ice-
cooled EDTA solution (20 mM). Then, 5 mL of the prepared ho-
mogenate was mixed with 4 mL of distilled water (4°C) and
1 mL of trichloroacetic acid (50% w/v). The mixture was centri-
fuged (10000 g, 4°C, 15 min). Then, 2 mL of the supernatant was
mixed with 4 mL of Tris buffer (0.4 M, pH = 8.9), and 100 μL of
Ellman’s reagent (DTNB, 0.01 M in methanol). Finally, the
absorbance of developed color was measured at 412 nm using
an Ultrospec 2000 UV spectrophotometer (Uppsala, Sweden).21
Ferric reducing antioxidant power (FRAP) of kidney
tissue
FRAP assay measures the formation of a blue colored Fe2+-
tripyridyltriazine compound from the colorless oxidized Fe3+
form by the action of electron-donating antioxidants.22 In the
current study, the working FRAP reagent was freshly prepared
by mixing 10 volumes of acetate buffer (300 mmol/L, pH 3.6),
with 1 volume of TPTZ (10 mmol/L in 40 mmol/L hydrochloric
acid) and 1 volume of ferric chloride (20 mmol/L). Kidney tis-
sue was homogenized in an ice-cooled Tris buffer (250 mM
Tris–HCl, 200 mM sucrose and 5 mM DTT, pH = 7.4). Then,
50 μL of tissue homogenate and 150 μL of deionized water
was added to 1.5 mL of the FRAP reagent. The reaction mixture
was incubated at 37°C for 5 min. Finally, the absorbance of de-
veloped color was measured at 595 nm by an Ultrospec 2000
UV spectrophotometer (Uppsala, Sweden).23
Kidney mitochondria isolation
Rat kidneys were washed and minced in an ice-cold buffer me-
dium (70 mM mannitol, 220 mM sucrose, 2 mM HEPES,
0.5 mM EGTA and 0.1% BSA, pH = 7.4). Minced tissue was
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Valproicacid-induced
Valproic acid-induced Fanconi syndrome
transported into a fresh buffer (5 mL buffer/1 g of the kidney)
and homogenized. Mitochondria were isolated by differential
centrifugation of the kidney homogenate.24 First, unbroken
cells and nuclei were pelleted at 1000 g for 10 min at 4°C; sec-
ond, the supernatant was centrifuged at 10000 g for 10 min at
4°C to pellet the mitochondria. This step was repeated three
times using fresh buffer medium. Final mitochondrial pellets
were suspended in a buffer containing 70 mM mannitol,
220 mM sucrose, 2 mM HEPES, and 0.5 mM EGTA, pH = 7.4,
except for the mitochondria used to assess ROS production, mi-
tochondrial depolarization and mitochondrial swelling, which
were suspended in respiration buffer (320 mM Sucrose,10 mM
Tris, 20 mM MOPS, 50 μM EGTA, 500 μM MgCl2, 0.1 mM
KH2PO4 and 5 mM Sodium succinate, pH = 7.2), MMP assay
buffer (220 mM Sucrose, 68 mM Mannitol, 10 mM KCl,
5 mM KH2PO4, 2 mM MgCl2, 50 μM EGTA, and 10 mM
HEPES, pH = 7.2) and swelling buffer (125 mM Sucrose,
65 mM KCl, 10 mM HEPES, pH = 7.2).24 Samples protein con-
centrations were determined by the Bradford method.25
Mitochondrial succinate dehydrogenase activity
(MTT assay)
The 3-(4, 5-dimethylthiazol-2-yl)-2, the 5-diphenyltetrazolium
bromide (MTT) assay was applied as a colorimetric method for
determination of mitochondrial succinate dehydrogenase ac-
tivity (SDA) as previously described.26,27 Briefly, a mitochon-
drial suspension (0.5 mg protein/mL) was incubated with
0.4% of MTT at 37°C for 30 min. The product of purple
formazan crystals was dissolved in 1 mL dimethyl sulfoxide
(DMSO) and the optical density (OD) at 570 nm was measured
with an EPOCH plate reader (Bio-Tek Instruments, Highland
Park, USA).
Mitochondrial depolarization
Mitochondrial uptake of the cationic fluorescent dye, rhoda-
mine 123, has been used for the estimation of mitochondrial
depolarization.24,28,29 Rhodamine 123, accumulates in intact
mitochondria by facilitated diffusion. When the mitochondrion
is damaged and depolarized, there is no facilitated diffusion and
the amount of rhodamine 123 in the supernatant is increased.
In the current investigation, the mitochondrial fractions
(0.5 mg protein/mL) were incubated with 10 μM of rhodamine
123 in the MMP assay buffer for 30 min. Afterward, samples
were centrifuged (15 000 g, 1 min, 4°C) and the fluorescence
intensity of the supernatant was monitored using a FLUOstar
Omega multifunctional microplate reader (λ excitation = 485 nm
and λ emission = 525 nm).24,30
Reactive oxygen species (ROS) in isolated kidney
mitochondria
20 , 70 -dichlorofluorescein diacetate (DCFH-DA) was used as a
fluorescent probe to assess mitochondrial ROS formation.
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Briefly, isolated kidney mitochondria were placed in the respi-
ration buffer. Then, 5 μL of DCFH-DA was added (Final con-
centration, 10 μM) and the mixture was incubated for 30 min
in the dark. The fluorescence intensity of samples was mea-
sured using a FLUOstar Omega multifunctional microplate
reader (λ excitation = 485 nm and λ emission = 525 nm).24,30
Mitochondrial swelling
Analysis of mitochondrial swelling was estimated through
changes in light scattering as monitored spectrophotometrically
at 540 nm (25°C).24,30 Briefly, isolated mitochondria were
suspended in swelling buffer and the absorbance was measured
at 540 nm during 70 min of incubation using an EPOCH plate
reader (Bio-Tek Instruments, Highland Park, USA). A decrease
in absorbance indicates an increase in mitochondrial
swelling.24,30
Mitochondrial glutathione (GSH) content
Mitochondrial GSH level was determined with a method using
Ellman reagent (5,5-dithiobis-2-nitrobenzoic acid, DTNB).31
The mitochondrial suspensions (0.5 mg/mL, in phosphate
buffer, pH = 7.4) were treated with 250 μL of trichloroacetic
acid (10% w/v) to extract mitochondrial glutathione. The mix-
ture was centrifuged (15000 g, 10 min, 4°C) to remove dena-
tured proteins. Afterward, 100 μL of DTNB (0.04% in
phosphate buffer) was added and the absorbance of samples
was recorded at 412 nm with an EPOCH plate reader (BioTek
Instruments, Highland Park, USA).24,27
Lipid peroxidation in isolated kidney mitochondria
Thiobarbituric acid-reactive substances (TBARS) test was used
for lipid peroxidation assay in isolated kidney mitochondria.24
As previously mentioned, sucrose interferes with the TBARS
assay.24 Hence, isolated mitochondria were washed once (to
remove sucrose) in ice-cooled MOPS-KCl buffer (50 mM
MOPS, 100 mM KCl, pH = 7.4), and re-suspended in MOPS–
KCl buffer.24 Afterward, the mitochondrial suspension was
added with twice its volume of a mixture containing trichloro-
acetic acid (15% w/v), thiobarbituric acid (0.375%), HCl
(0.24 N) and Trolox (0.5 mM). Samples were heated for
15 min at 100°C.24 After centrifugation (15 000 g, 10 min),
the absorbance of the supernatant was measured at 532 nm
using an EPOCH plate reader (BioTek Instruments, Highland
Park, USA).24
Mitochondrial ATP level
Based on a previously reported procedure, mitochondrial ATP
level was assessed by HPLC.32 Briefly, isolated mitochondria
(0.5 mg protein/mL) were treated with ice-cooled 0.2 M
perchloric acid and centrifuged (10 min, 13000, rpm, 4°C).
Afterward, the supernatant (100 μL) was treated with its
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equivalent volume of ice-cooled 1 M KOH solution. Samples
(60 μL) were injected into an HPLC system consisted of an
LC-18 column (μ-Bondapak, 15 cm). The mobile phase was
composed of KH2PO4 (215 mM), tertiary butyl ammonium sul-
fate (2.3 mM), KOH (1 M, 0.4%) and acetonitrile (4%). The
flow rate was 1 mL/min and the UV detector in this system
was set at 254 nm.32 There was a good linear relationship
among standard ATP concentrations at a range of 0.0–0.6 μM
against its peak area with correlation coefficient being 0.9944.
Statistical analysis
Data are given as the Mean ± SEM. Data comparison was per-
formed by the one-way analysis of variance (ANOVA) with
Tukey’s multiple comparison test as a post hoc. P < 0.05 was
considered significant.
RESULTS
Valproic acid-treated animals developed biochemical evidence
of renal injury and FS (Table 1). Elevated serum GGT, ALP,
Cr, and BUN along with hypokalaemia, hypophosphataemia,
and a decrease in serum uric acid, were detected in VPA-treated
animals (Table 1). Administration of carnitine significantly de-
creased serum biomarkers of renal injury in VPA group
(Table 1). There was no significant difference in almost all the
parameters assessed when two doses of VPA (250 and
500 mg/kg, i.p) were compared. Although the effect of VPA
on the biochemical markers of kidney injury doesn’t seem to
be dose-dependent, several biochemical parameters were sig-
nificantly higher in VPA 500 mg/kg treated group in compari-
son with VPA 250 mg/kg (Table 1). Previous investigations
also mentioned 500 mg/kg of VPA as the nephrotoxic dose of
this drug.6,15,33
The level of oxidative stress markers in kidney was signifi-
cantly changed in VPA-treated animals (Fig. 1). It was found
that VPA caused an increase in kidney ROS formation and lipid
peroxidation (Fig. 1). Moreover, renal glutathione reservoirs
were depleted and tissue antioxidant capacity was defected in
VPA-treated rats (Fig. 1). Carnitine (100 mg/kg) significantly
increased GSH levels and the antioxidant capacity of kidney tis-
sue in VPA-treated animals (Fig. 1). Moreover, kidney ROS and
LPO levels were significantly decreased when VPA-treated rats
received carnitine (100 mg/kg, i.p.) (Fig. 1).
Histopathological presentation of VPA-induced renal injury
included interstitial nephritis, tissue necrosis, and atrophy
(Fig. 2B, C and Table 2). The histopathological changes in
VPA-treated animals at a dose of 250 mg/kg, were evident as
mild interstitial nephritis (Fig. 2, D). It was found that carnitine
(100 mg/kg) hampered renal tissue injury induced by VPA
(500 mg/kg, i.p.) (Fig. 2, E). We found no sign of kidney tissue
fibrosis in VPA-treated animals (Masson trichrome staining)
when it was compared with control (vehicle-treated) group
(Fig. 3). Liver histopathological changes including steatosis,
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Valproic acid-induced Fanconi syndrome

Table 1 Serum biochemical measurements

Control VPA VPA VPA 500 mg/kg +

250 mg/kg 500 mg/kg Carnitine 100 mg/kg
,
ALP (U/L) 1140 ± 350 2740 ± 139* 7367 ± 2653* a 1018 ± 109b
γ-GT (U/L) 11 ± 1 29 ± 5* 42 ± 7* 11 ± 2b
Creatinine (Cr) 0.67 ± 0.07 0.54 ± 0.15 0.66 ± 0.06 0.66 ± 0.1
,
BUN (mg/dL) 59 ± 17 90 ± 10 155 ± 27* a 57 ± 9b
,
BUN/Cr Ratio 90 ± 17 176 ± 50 242 ± 41* a 88 ± 18b
2+
Ca (mg/dL) 5.04 ± 0.15 4.2 ± 0.4 4.02 ± 0.25 4.93 ± 0.11
+
K (mmol/L) 5.6 ± 1.6 3.54 ± 1.1* 3.61 ± 0.53* 4.46 ± 0.43b
+
Na (mmol/L) 78.4 ± 4.3 76 ± 4 69 ± 2.9 70.6 ± 7.8
Glucose (mg/dL) 103 ± 6 112 ± 2 90 ± 5 119 ± 8
,
Uric acid (mg/dL) 1.6 ± 0.13 0.42 ± 0.02* 0.52 ± 0.03* a 1.08 ± 0.03b
Phosphate (mg/dL) 2.9 ± 0.12 2.32 ± 0.23 1.77 ± 0.27* 2.15 ± 0.10b
Total protein (mg/dL) 7.19 ± 0.8 6.12 ± 0.5 7.13 ± 0.4 7.10 ± 0.4

Data are given as Mean ± SD (n = 8). VPA, Valproic acid. *Indicates significantly different as compared with control animals (P < 0.01). aIndicates significantly different as
compared with VPA (250 mg/kg)-treated group (P < 0.01). bIndicates significantly different as compared with VPA (500 mg/kg)-treated group (P < 0.01).

Fig. 1 Kidney tissue reactive oxygen species (ROS)

formation, lipid peroxidation, glutathione content, and
total antioxidant capacity. Data are shown as
Mean ± SD (n = 8). In carnitine-treated group, animals
received valproic acid (VPA) (500 mg/kg per day) plus
carnitine (100 mg/kg per day) for 15 consecutive days.
*Indicates significantly different as compared with
a
control group (P < 0.001). Indicates significantly
different as compared with VPA-treated animals
ns
(P < 0.001). : not significant as compared with
control group. Carn, carnitine.

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Fig. 2 Photomicrographs of kidney histopathology in valproic acid-treated animals. Kidney photomicrographs showed interstitial nephritis (Black arrow), and tissue
necrosis (Yellow arrow), in valproic acid (VPA) (500 mg/kg)-treated animals (B and C) in comparison with control group (A). VPA 250 mg/kg showed mild interstitial
nephritis (D). No significant histopathological changes were detected in carnitine-treated animals (E; VAP 500 mg/kg + Carnitine 100 mg/kg). Scale bar, 1000 μm.

increased mitochondrial lipid peroxidation (Fig. 5). We found

Table 2 Kidney histopathological changes in valproic acid-treated rats
that carnitine (100 mg/kg) co-administration to VPA-treated
Treatment Focal Tubular Interstitial Vascular rats, abrogated VPA-induced mitochondrial dysfunction in the
Necrosis Atrophy Inflammation congestion
kidney (Figs 4 and 5).
Control (Vehicle-treated) 0 0 0 0
VPA 500 mg/kg 0–1 0–1 1 0–1
VPA 250 mg/kg 0 0 0–1 0–1
VPA 500 mg/ 0 0 0–1 0 DISCUSSION
kg + Carnitine 100 mg/kg
There is evidence of kidney injury in patients treated with
VPA, Valproic acid.
VPA.3,5,8,34–36 However, the mechanism of VPA-induced renal
dysfunction remains obscure. In the current investigation we
have demonstrated mitochondrial dysfunction and increased
necrosis, and inflammation were also detected in VPA-treated oxidative stress in the kidney.
groups (Data not shown). Oxidative stress constitutes an important risk factor for tissue
The effect of VPA was not dose-dependent at the mitochon- damage and organ dysfunction. Oxidative stress is involved in
drial level. It was found that mitochondria isolated from VPA- the activation of several signalling pathways, leading to the ac-
treated animals showed marked decrease in succinate tivation of transcription factors, gene expression, and induction
dehydrogenase (SDA) activity (MTT assay) (Fig. 4). Further as- of apoptosis.37 Toxicity of VPA on renal tubular cells has been
sessment of kidney mitochondria, derived from VPA-treated demonstrated both in vitro and in vivo.38–40 Renal tubular disor-
groups, revealed a marked increase in mitochondrial swelling, der and increased levels of BUN, Cr, and kidney tissue histo-
mitochondrial depolarization, and an increase in mitochondrial pathological changes are detected in VPA-treated animals.6,9
ROS level (Fig. 4). It was also detected that mitochondrial glu- VPA inactivates antioxidant enzymes.41,42 Our results are in
tathione stores and ATP level were decreased in VPA-treated consensus with the other investigations, which quoted that
animals (Fig. 5). VPA administration (500 mg/kg) also oxidative stress developed in renal tissue after VPA

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Fig. 3 Kidney fibrosis in valproic acid-treated rats. (A) (Control group; Vehicle-treated) and (B) (VPA 500 mg/kg per day, i.p., for 14 days). VPA-treated group (B) showed
no change in tissue collagen deposition compared to the vehicle-treated group (A). Scale bar, 500 μm.
administration.6,12,15,33 We found that VPA elevated kidney
ROS levels, reduced tissue antioxidant activity, increased lipid
peroxidation and depleted renal glutathione stores. Moreover,
we found that renal mitochondrial function was hampered in
VPA-treated animals. All these data indicate the contribution
of oxidative stress, mitochondrial dysfunction, and its conse-
quent events in VPA-induced renal injury and FS. Oxidative
stress might be a cause or a consequence of mitochondrial
dysfunction. Hence, VPA-induced ROS formation might deteri-
orate mitochondrial injury and vice versa.
In the current investigation, we found no sign of tissue fibro-
sis in the kidney of VPA-treated animals as assessed by Masson
trichrome staining (Fig. 3). VPA may cause kidney fibrosis at
other doses or after longer administration.
The molecular mechanisms controlling proximal tubule
reabsorption of proteins, ions, and other solutes have well been
elucidated. On the other hand, FS is characterized by multiple
transport defects in the kidney.10 Impaired basolateral sodium
and potassium adenosine triphosphate (Na+, K+-ATPase) activ-
ity is the physiological characteristic of the FS.8,10 Renal proxi-
mal tubules have a high metabolic rate and generate more than
95% of their ATP by means of oxidative phosphorylation.43
Proximal tubular cells are highly sensitive to low ATP and en-
ergy crisis.43,44 Energy metabolism disorders can affect almost
any organ. In the kidney, ions and electrolytes reabsorption is
an energy-dependent process. Na+/K+ ATPase pump consumes
ATP molecules to reabsorb ions and electrolytes into the
blood.45 High and constant dependence of proximal tubular
cells on energy highlights the importance of mitochondria.
The effect of VPA on kidney mitochondria has not been
investigated so far. In the current study, we found that kidney
mitochondrial ATP level significantly dropped in VPA-treated
animals. Hence, VPA-induced mitochondrial dysfunction
might lead to the energy crisis in renal proximal tubule cells.
Valproic acid-induced mitochondrial toxicity has been
shown in the liver.11 Adverse mitochondrial events associated
© 2017
© 2017 Asian
Asian Pacifi
Pacific Society of Nephrology
c Society Nephrology
Valproicacid-induced
Valproic acid-induced Fanconi syndrome
with VPA in the liver include inhibition and decreased the
activity of mitochondrial complexes Ι and V, inhibition of oxy-
gen consumption and ATP synthesis, impaired inner mitochon-
drial membrane matrix organization, inhibition of
mitochondrial β-oxidation, impaired mitochondrial oxidative
phosphorylation, and vacuolar fragmentation.11–13 In the cur-
rent investigation, we found that kidney mitochondria might
also be a potential target for VPA to induce kidney injury and FS.
Discontinuation of VPA may cause remission of symptoms
within weeks but can take up to months and even years.4,36
Hence, antioxidants and mitochondrial protective agents might
prevent injury or enhance the remission of patients with VPA-
induced FS.
Carnitine has been recognized to possess a variety of bio-
logical and pharmacological activities, including a cofactor
for the transportation of free fatty acid from cytosol to the mi-
tochondria.46 Carnitine also acts as an antioxidant and radical
scavenger.47 Recent research has shown that some patholog-
ical conditions benefit from exogenous carnitine supplemen-
tation.48–51 The protective effect of carnitine in different
models of renal injury has also been reported.52–54 Supple-
mentation with carnitine offers beneficial effects in patients
with VPA-induced liver injury.14 It has been shown that car-
nitine showed a good safety profile in patients with acute
VPA overdose.55,56 In the current study, we found that carni-
tine effectively mitigated VPA-induced renal injury and mito-
chondrial dysfunction. Carnitine (100 mg/kg, i.p.) along with
VPA, significantly restored tissue antioxidant capacity and
effectively encountered VPA-induced oxidative stress in the
kidney (Fig. 1). Furthermore, mitochondria isolated from
the kidney of the carnitine-treated group had better function
(Figs 4 and 5). The antioxidant and radical scavenging proper-
ties of carnitine might be involved in this process. As risk
assessment investigations revealed a safe profile for carnitine
in humans,57 carnitine might have therapeutic potential
against VPA-induced FS.
357
Heidari
R R et
Heidari et al.
al.
Interestingly, some investigators have shown that VPA has
renoprotective properties.58–60 It has been reported that VPA
prevented focal segmental glomerulosclerosis, proteinuria,
and kidney fibrosis in a mice model.58,60 VPA, as an effective
inhibitor of histone deacetylase (HDAC), serves as an anti-
inflammatory and anti-fibrotic agent.58,60 Although a lower
dose VPA acts as a renoprotective agent,58,60 a higher dose of
VPA was administered here to simulate VPA-induced renal
injury in rats.6,43
The insignificant changes in serum Cr and fibrosis in the cur-
rent study might reflect the mild kidney injury in VPA-treated
rats (Table 1). This might also be due to the duration of expo-
sure to VPA. On the other hand, the occurrence of oxidative
stress and mitochondrial dysfunction in VPA-treated animals
might reflect the early signs of VPA-induced nephrotoxicity.
358
Fig. 4 Kidney mitochondrial swelling, reactive oxygen
species (ROS) formation, MTT assay, and mitochondrial
depolarization in control and VPA-treated animals.
Carn, carnitine; VPA, valproic acid. Data are given as
ns
Mean ± SD (n = 8). not significant as compared with
control. Asterisks indicate significantly different as
compared with control (**P < 0.01, and ***
a
P < 0.001). Indicates significantly different as
compared with VPA (500 mg/kg)-treated group
(P < 0.001).
Other factors such as dehydration, high protein diet and also
VPA-induced hepatic injury (which is concurrently occurs in
VPA-treated animals) could also affect serum biomarkers of
VPA-induced renal damage (e.g. BUN). Hence, monitoring the
urine analytical parameters and glomerular/tubular function,
as well as more precise biomarkers of renal injury could help
monitoring VPA-induced nephrotoxicity in animal models
and/or patients.
Previous studies mentioned the dose of 500 mg/kg, i.p for 14
consecutive days as the nephrotoxic dose of VPA.6,16,34 Our in-
vestigation also revealed the renal injury, oxidative stress, and
mitochondrial dysfunction when animals received VPA
500 mg/kg. Hence, VPA could be applicable as an animal model
of FS. However, based on the insignificant change in some bio-
markers of renal injury (e.g. Cr), we suggest a longer time of
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© 2017Asian
AsianPacifi
Pacific Society of
c Society of Nephrology
Nephrology

Fig. 5 Mitochondrial glutathione content, lipid peroxidation and ATP level in the kidney of VPA-treated rats. Data are given as Mean ± SD (n = 8). ns: not significant as
compared with control. Asterisks indicate significantly different as compared with control (*P < 0.05, *** P < 0.001). Indicates significantly different as compared with
VPA 500 mg/kg (P < 0.001).
drug administration for a more reliable animal model of VPA-
induced FS.
Several investigations indicate the role of reactive metabo-
lites in VPA-induced cytotoxicity, organelle dysfunction, and
oxidative stress.61,62 There is no study on VPA biotransforma-
tion and potential toxic intermediates formation in the kidney.
Hence, future investigations might shed light on the role of
drug biotransformation in the mechanism of VPA-induced
mitochondrial dysfunction, renal injury, and FS.
Current data might provide insights to determine the
mechanisms responsible for the VPA-induced renal injury.
Based on this investigation, we propose that prexisting
mitochondrial defects might exacerbate VPA-induced FS.
Moreover, these data suggest carnitine as a potential
nephroprotective agent against VPA-induced renal injury. On
the other hand, measuring VPA level in the kidney tissue, as
well as in the mitochondrial preparations might help to further
characterization of VPA-induced FS and mitochondrial dys-
function. Moreover, determination of urine analytical parame-
ters and glomerular/tubular function could greatly enhance
our understanding of the magnitude of injury induced by FS-
inducing xenobiotics.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.
ACKNOWLEDGEMENTS
This investigation was financially supported by the Vice Chan-
cellor of Research Affairs of Shiraz University of Medical
© 2017
© 2017 Asian
Asian Pacifi
Pacific Society of Nephrology
c Society Nephrology
Valproicacid-induced
Valproic acid-induced Fanconi syndrome
b
Sciences (Grant number: 94-01-36-10650). Authors thank
Pharmaceutical Sciences Research Center (PSRC) of Shiraz
University of Medical Sciences for providing technical facilities
to carry out this study. The authors also wish to offer very spe-
cial thanks to Professor Negar Azarpira (Transplant Research
Center, Shiraz University of Medical Sciences, Shiraz, Iran)
for her kind revision of the histopathological findings of the
current investigation.
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