Department of Biochemistry, Panjab University, Chandigarh 160 014, IndiaDepartment of Bioinformatics, D.A.V. College, Chandigarh 160 014, IndiaCenter with Potential for Excellence in Biomedical Sciences, Panjab University, Chandigarh 160 014, IndiaBiotechnology Branch, University Institute of Engineering and Technology, Panjab University, Chandigarh 160 014, India
r t i c l e i n f o
rticle history:eceived 5 May 2010eceived in revised form 20 July 2010ccepted 15 August 2010
eywords:olic acidcute renal failure (ARF)ipid peroxidationlutathionecanning electron microscopy (SEM)
a b s t r a c t
Systemic administration of folic acid (FA) in mice was used for studying the pathogenesis associatedwith acute renal failure (ARF). However, the mechanism by which FA induces ARF remains poorly under-stood. The present study therefore, was planned to investigate the effect of folic acid administration onprooxidant state and associated ultrastructural changes in renal tissue. Balb/c male mice of 4–6 weeksold were divided into control and two folic acid treatment groups (Groups A and B). The animals in groupA were administered intraperitoneal injection of folic acid (100 mg kg−1 body weight) for a period of 7consecutive days while the animal in group B were administered a single intraperitoneal dose of folic acid(250 mg kg−1 body weight). The renal tissues were collected and used for the analyses of lipid peroxida-tive indices and activities of antioxidant enzymes in renal tissues. To corroborate biochemical findingsscanning electron microscopy (SEM) in renal tissue was studied. Folic acid treated animals demonstratedmarked renal hypertrophy accompanied by severe impairment of renal function. Glutathione levels (GSH)and antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px)
levels were significantly decreased and LPO levels increased following FA treatment. SEM results furthersubstantiated the observed biochemical changes as evident by severe inflammation in glomeruli, swellingin primary and secondary pedicels, blebbing in villi, and tremendous deprivation of erythrocytes (RBCs)in FA treated kidneys. The present study therefore suggests that acute administration of folic acid leads tothe generation of oxidative stress and altered membrane architecture responsible for folic acid inducedARF.
Folic acid (FA) is one of the model compound commonly usedo study the pathophysiology associated with acute renal failureARF) (Cheng et al., 2005; Ortega et al., 2005; Szczypka et al., 2005;iaschi-Taesch et al., 2004). Variety of nephrotoxins viz. mercury,entamicin, glycerol, cisplatin, cyclosporine A is known to causeRF (Baliga et al., 1999). As a member of Vitamin B complex, it isenerally needed for cell replication and growth and helps form-ng building blocks of DNA, RNA. High concentration of folic acidas been found to be toxic to the various organs of the body espe-ially kidney. Folic acid induced acute renal failure (FA-ARF) isssociated with the rapid appearance of folic acid crystals within
he renal tubules and subsequent acute tubular necrosis, followedy epithelial regeneration and renal cortical scarring (Bosch et al.,993; Mullin et al., 1976). FA-ARF is characterized by tubular injury,
including tubular cell apoptosis, as well as tubular cell prolifera-tion, inflammatory cell infiltration, and mild fibrosis in the chronicphase (Ortega et al., 2005; Doi et al., 2006; Fang et al., 2005; Dai etal., 2002; Ortiz et al., 2000). Interestingly, all these features are alsofound in human ARF, suggesting that FA-ARF is an excellent modelthat mimics human ARF. But little is known of the mechanism(s)by which folic acid mediates its toxicity and its effect on the renalprooxidant state.
The involvement of oxidative stress in the progression in renalinjury has been recognized a key player in pathophysiologic path-ways of a wide variety of progressive and experimental renaldiseases (Haugen and Nath, 1999). Kidney has a very active oxida-tive metabolism because of its transport function which resultsin the production of reactive oxygen species (ROS), which leftunchecked, can damage all major cellular components and leadto a state of oxidative stress (Maser et al., 2002). Therefore, the
present study was designed to investigate the effect of folic acidtreatment under acute and short-term administration in mice renalprooxidant state. The study presented here demonstrates that fol-lowing acute administration of folic acid induces a prooxidant state
Table 1Effect of folic acid administration on body weights in mice.
Body weight (g)
ControlGroup A 7 days 29 ± 2
20 ± 3*** (−31%)Group B 12 h 27 ± 2 (−6.89%)
24 h 26 ± 3 (−10.34%)
*
iio
2
2
Uc
2
cUblslov
Fa*
3.2. Sample preparation and biochemical assays
Animals were euthanized post-dosing (7 days in group A and 12,24 and 36 h interval in group B after the last injection) by cervical
36 h 27 ± 2 (−6.89%)
P < 0.05, **P < 0.01, ***P < 0.001 as compared to control (student t-test); n = 6.
n renal tissue by increasing lipid peroxidation and reduced antiox-dant enzyme protection which could be the key step in initiationf ARF.
. Materials and methods
.1. Chemicals
Folic acid was purchased from Sigma–Aldrich (St Louis, MO,SA). All other chemicals used were of analytical grade and pur-hased from local Sigma–Aldrich.
.2. Animals and their treatment
Male balb/c mice of 4–6 weeks old weighing 20–30 g were pur-hased from a colony raised in the Central Animal House, Panjabniversity, Chandigarh, India. The animals were housed on huskedding in clean polypropylene cages and were fed Hind Lever pel-
et diet and water ad libitum. The protocols used in the study weretrictly according to the guidelines on the human use and care of
aboratory animals and were approved by the ethical committeef the Panjab University. The mice were randomly segregated intoarious groups with each group having six animals.
ig. 1. Effect of folic acid administration on (a) serum blood urea nitrogen (BUN)nd (b) serum creatinine in group A (100 mg/kg i.p.) and group B (250 mg/kg i.p.).P < 0.05, **P < 0.01, ***P < 0.001 as compared to control (student t-test); n = 6.
ogic Pathology 64 (2012) 225– 232
3. Experimental design
Animals were divided into two groups, control (C) and folic acidtreatment group. FA treated group was further subdivided intotwo groups, group A and group B. The animals in group A wereadministered intraperitoneal injection of folic acid (100 mg kg−1
body weight) for a period of seven (7) consecutive days, and weresacrificed on day 8th. The animals in group B were administered asingle intraperitoneal dose of folic acid (250 mg kg−1 body weight)and were sacrificed after 12, 24 and 36 h intervals. Folic acidwas dissolved freshly in double distilled water each time beforeadministration. Body weights were also measured every day beforeadministering folic acid. These doses of folic acid though inducednephrotoxicity but were also associated with a morbidity rate of<5% for the experimental period.
3.1. Renal function
To carry out renal function studies the serum was prepared aftercollecting the blood samples through the cardiac puncture of theanimal following overnight fasting. Renal function was assessed bymeasuring the levels of creatinine and blood urea nitrogen (BUN)using a commercially available kit (Reckon Diagnostics Pvt Ltd, Bar-oda, INDIA). The level of serum creatinine and BUN was expressedas milligrams per 100 ml (mg/dl).
Fig. 2. Effect of folic acid administration on (a) GSH levels and (b) MDA levels ingroup A (100 mg/kg i.p.) and group B (250 mg/kg i.p.) in mice kidney homogenate.*P < 0.05, **P < 0.01, ***P < 0.001 as compared to control (student t-test); n = 6.
A. Gupta et al. / Experimental and Toxicologic Pathology 64 (2012) 225– 232 227
Table 2Effect of folic acid administration on kidney weights and kidney/body weight ratio.
Avg. kidney weight (g) Kidney/body weight ratio (%)
Control 0.1765 ± 0.0319 0.60 ± 0.06Group A 7 days 0.2524 ± 0.0145*** (+42.82%) 1.26 ± 0.21*** (+110%)Group B 12 h 0.2289 ± 0.0088* (+29.68%) 0.84 ± 0.18* (+40%)
24 h 0.2008 ± 0.0176 (+13.76%) 0.77 ± 0.26 (+28.33%)0.0228
*
dstsmt
3
loeFswtwpt
Fa
36 h 0.2144 ±
P < 0.05, **P < 0.01, ***P < 0.001 as compared to control (student t-test); n = 6.
islocation; their kidneys were removed, rinsed in ice-cold isotonicaline. The kidneys were blotted, dried and weighed. A 10% (w/v)issue homogenate was prepared in 50 mM phosphate bufferedaline (pH 7.4) using Potter-Elvehjem-type glass homogenizer. Postitochondrial supernatant (PMS) was prepared by differential cen-
rifugation.
.3. Biochemical estimations
Lipid peroxidation (LPO) was estimated by measuring the MDAevels by thiobarbituric acid reactive substances (TBARS) methodf Ohkawa et al. (1979). Reduced glutathione (GSH) content wasstimated in the homogenate by the method of Roberts andrancetic (1993). Superoxide dismutase (SOD) activity was mea-ured by the method of Kono (1978). One unit of SOD activityas expressed as the enzyme protein amount causing 50% inhibi-
ion in nitroblue tetrazolium reduction rate. Catalase (CAT) activityas measured by measuring the absorbance decrease of hydrogeneroxide (H2O2) at 240 nm by the method of Aebi (1984). Glu-athione peroxidase (GSH-Px) activity was measured by following
ig. 3. Effect of folic acid administration on (a) superoxide dismutase (SOD), (b) catalasend group B (250 mg/kg i.p.) in mice kidney post mitochondrial supernatant. *P < 0.05, **P
* (+21.47%) 0.79 ± 0.14* (+31.67%)
changes in NADPH absorbance at 340 nm by the method of Floheand Gunzler (1984). Glutathione-S-transferase (GST) activity wasassayed as described by the method of Warholm et al. (1985). Pro-tein content was estimated according to the method of Lowry et al.(1951). BSA was used as a standard.
3.4. Scanning electron microscopy (SEM)
For scanning electron microscopic examination, kidneys werecut and fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH7.4) for 2 h, dehydrated with a series of ethanol (50–100%) and driedusing critical point drier. After drying the samples were mountedon an aluminium stub using silver paint. Samples were then intro-duced into the chamber of sputter coater and coated with gold. SEMJEOL was used for viewing and photographing.
3.5. Statistical analysis
All assays were performed in triplicate. Data reported asmean ± standard error of mean of six animals per group. Valueswith P < 0.05 were considered as statistically significant.
(CAT) and (c) glutathione peroxidase (GSH-Px) activity in group A (100 mg/kg i.p.) < 0.01, ***P < 0.001 as compared to control (student t-test); n = 6.
228 A. Gupta et al. / Experimental and Toxicol
Fig. 4. Effect of folic acid administration on glutathione-S-transferase (GST) activ-im(
4
crgcm
Fgw
indicated by reduced glutathione content and elevated LPO levels
ty in group A (100 mg/kg i.p.) and group B (250 mg/kg i.p.) in mice kidney postitochondrial supernatant. *P < 0.05, **P < 0.01, ***P < 0.001 as compared to control
student t-test); n = 6.
. Results
The acute administration of folic acid produced significanthanges in the kidney/body weight ratio along with impaired
enal function. At the end of the experiment, body weights inroup A were 31% less than the control animals, however thehanges observed in the body weights of animals in group B wasarginal (Table 1). Table 2 illustrates kidney/body weight ratio
ig. 5. (a) Intact kidney glomerulus of control group (SEM ×1000) and (b) kidneylomerulus in folic acid treated group A, swelling in podocytes can be seen due tohich urinary space around the glomerulus appear crowded (SEM ×1000).
ogic Pathology 64 (2012) 225– 232
in groups A and B which increased tremendously by 110% ingroup A manifesting renal hypertrophy whereas in group B maxi-mum increase observed was approximately 40% (12 h). There wassevere impairment of renal function as observed by significantelevation in serum BUN and creatinine levels following folic acidtreatment. Clinical signs of ARF in mice, such as fatigue, reducedalertness and bristling of the coat were also observed after folicacid administration. Serum BUN levels in group A was significantlyincreased (P < 0.05) by 351% while group B observed maximumincrease by 254% (24 h) (Fig. 1a). Similarly, serum creatinine lev-els were elevated significantly (P < 0.05) by 202% (group A) whilegroup B observed increase by 156% (12 h) (Fig. 1b). Various reportshave implicated impaired renal function along with generation ofoxidative stress in acute renal injury models. To assess the renalprooxidant state, lipid peroxidation, glutathione content and enzy-matic activity of antioxidant enzymes were determined followingfolic acid treatment. Folic acid administration resulted in signifi-cant decrease (P < 0.05) in glutathione content by (22.55%) in groupA while group B observed maximum decrease by 25.09% (24 h)(Fig. 2a). To corroborate these findings it was found that LPO levelswere significantly increased (P < 0.05) in group A by 29.51% whilein group B the LPO levels were elevated by 14.7% (12 h), 20.84%(24 h), and 12.18% (36 h) (Fig. 2b). This increased prooxidant state
following folic acid administration were further substantiated bydecreased activities of antioxidant enzymes SOD, CAT and GSH-Px. Group A observed significant decrease (P < 0.05) in the SOD
Fig. 6. (a) Kidney glomerulus of control group animals was embedded with normalshaped erythrocytes (RBCs) (SEM ×1300); (b) kidney glomerulus in folic acid treatedgroup A showing distorted shapes of erythrocytes which were not typical biconcavediscs, under normal blood flow conditions (SEM ×2500).
A. Gupta et al. / Experimental and Toxicologic Pathology 64 (2012) 225– 232 229
F tes weg ated gi
a(ddBnrara
ssatcbtepmniapewn
ig. 7. (a) At higher magnification, the surface contours of foot processes of podocyroup (SEM ×16,000); (b) interdigitating foot processes of podocytes in folic acid trenflammation among the foot processes and distorted knob-like microprojections.
ctivity by 32.25% while group B observed reduction up to 25.60%36 h) (Fig. 3a). The reduction in SOD activity was accompanied byecreased activities of CAT and GSH-Px. Group A observed 15.99%ecrease in CAT activity while there was marginal decrease in group
(Fig. 3b). GSH-Px activity was also found to be decreased sig-ificantly (P < 0.05) by (23.87%) in group A while in group B theeduction was approximately 19.07% (36 h) (Fig. 3c). Although GSTctivity was decreased in group A (23.02%) (Fig. 4) but group Bemained refractory to the changes in GST activity after folic aciddministration.
To examine the ultra structural changes in the renal tissue,canning electron microscopy (SEM) was performed. SEM revealedignificant difference in the morphology of renal structures in foliccid treatment groups compared to control. Folic acid administra-ion changed the morphology of podocytes dramatically, one of theommon pathological alteration of ARF. Most of the podocyte cellodies in glomerulus of group A became very swollen as comparedo control animals (Fig. 5a and b). SEM micrographs of erythrocytesmbedded in glomeruli of control animals (Fig. 6a) appeared com-letely normal while in group A, the erythrocytes showed distortedorphology (Fig. 6b), which were not typical biconcave discs, under
ormal blood flow conditions. Moreover, tremendous deprivationn the number of erythrocytes was observed in the glomeruli of foliccid treated kidneys. Fig. 7a represented the interdigitating foot
rocesses of podocytes in the animals of control group which cov-rs outer capillary surfaces. The surface contours of foot processesere smooth and tightly apposed each other, which is typical inormal functioning kidneys where as in folic acid treated animals
re smooth, tightly apposed each other and filtration slits were also narrow, controlroup A (SEM ×15,000) and (c) group B (12 h) (SEM ×14,000) showing swelling and
of group A and group B (12 h) swelling and inflammation betweenthe contours of foot processes was observed (Fig. 7b and c). The sur-face contours of foot processes in folic acid treated animals of groupA and group B (12 h) appeared flattened and distorted knob-likemicro projections was visible when compared to the foot processesof control group. Proximal convoluted tubules in the animals ofgroup A revealed considerable loss of brush border in responseto acute administration of folic acid (Fig. 8a). At higher magnifi-cation, severe blebbing of villi was observed in group A indicatingsigns of nephrotoxicity and apoptosis (Fig. 8b). However, no suchchange was visible in the animals of control group. Blebbing of villiin loop of henle was also evident in the animals of group B (12 h)(Fig. 8c). Moreover, tubular obstruction was observed by precipita-tion of large deposits of folic acid crystals in group A (Fig. 9a) andgroup B (36 h) (Fig. 9b) where as no such deposits was observed incontrol group.
5. Discussion
The study presented here establishes a relationship betweenincreased prooxidant state and acute renal injury followed by folicacid treatment. The inability of the animal to handle such highdoses of folic acid severely impairs its physiological response as
observed by significant reduction in food intake along with musclefatigue, reduced alertness and bristling of the coat. These signs aresuggestive of folic acid induced acute renal failure (FA-ARF) whichresulted in decreased body weight of the animals. The decrease in
230 A. Gupta et al. / Experimental and Toxicologic Pathology 64 (2012) 225– 232
Fig. 8. (a) Proximal convoluted tubules in folic acid treated group A animals which had undergone considerable brush border degeneration in response to folic acid inducedr arly sd thin l×
tletrce1aiocpsretvwtormegau
enal toxicity (SEM ×4000); (b) at higher magnification, blebbing of villi could be cleegeneration and blebbing of villi was also distinctly visible in the area around the600).
he body weight is in accordance with the reports in literature fol-owing acute administration of folic acid (Nabae et al., 2006; Want al., 2006; Tsutsumi et al., 2004). Folic acid administration ledo renal hypertrophy indicated by increased kidney/body weightatio. It is well documented in the literature that folic acid in highoncentrations is one of the most potent stimuli for cell prolif-ration in the rodent kidney (Baserga et al., 1968; Klinger et al.,980). In earlier experiments, increase in wet kidney weight wasssociated with a smaller increase in dry weight and a markedncrease in nucleic acid synthesis (Threlfall, 1968). Following lossf functional renal mass due to folic acid induced acute renal injuryould result in compensatory hypertrophy or regenerative hyper-lasia in mice kidney. The data on renal function confirmed thathort-term as well as acute administration of folic acid inducesenal dysfunction. Serum creatinine and BUN, indicative biomark-rs for kidney dysfunction were significantly elevated in folic acidreatment group A. In group B, serum creatinine and BUN were ele-ated after 12 h administration and thereafter started to decline,hich indicates a tendency of enhanced recovery of renal func-
ion. Dai et al. (2002) have reported similar finding that high dosef folic acid administration in mice induced ARF characterized byapid decline of renal function, tubular epithelial cell death andorphologic abnormalities. Over the last decade, there is growing
vidence for a role of reactive oxygen metabolites in the patho-enesis of a variety of renal diseases including gentamicin, glycerolnd cyclosporine A models of ARF. Therefore, it is imperative tonderstand the role of prooxidant/antioxidant status in the patho-
een in the folic acid treated animals of group A (SEM ×10,000) and (c) brush borderoop of Henle and tubular space in folic acid treated animals of Group B (24 h) (SEM
physiology associated with these renal injury models. Oxidativestress has been implicated in the pathogenesis of ARF in animalmodels (Baud and Ardaillou, 1986; Paller et al., 1984). GSH, animportant component of antioxidant defense mechanism, plays animportant role in protecting cells from xenobiotic-induced tissueinjury (Wu et al., 2004). The reduced levels of GSH in the folic acidadministered mice could be due to increased participation of GSHas an antioxidant in terminating free radicals produced by acuteadministration of folic acid. Depleted GSH levels have been welldocumented in animal as well as human ARF models suggesting anincreased prooxidant state in the renal tissue during experimen-tal ARF (Polat et al., 2006; Metnitz et al., 2000). Glutathione levelswere considerably depleted in folic acid treatment groups A and B.However, in group B depletion of GSH levels after 12 h and 24 h folicacid administration, there was a slight increase in the glutathionelevels after 36 h administration, indicating an adaptive response bythe kidney. The decrease in GSH levels might diminish the over-all antioxidant potential of the kidney resulting in increased LPOfollowing acute administration of folic acid. Lipid peroxidation isimplicated as the initial event in experimental ARF (Joannidis et al.,1989). Membrane lipids are a frequent target of ROS and can beoxidized to form lipid hydro peroxides. These lipid peroxides canundergo further reactions to produce various aldehydes, including
MDA, if not reduced by antioxidant enzymes such as glutathioneperoxidase (Esterbauer et al., 1991). The LPO levels were signifi-cantly increased in group A treated with short-term administrationof folic acid while in group B after initial increase at 12 h and 24 h
A. Gupta et al. / Experimental and Toxicol
Fig. 9. (a) Large deposits of precipitated folic acid crystals were observed underSEM in folic acid treated animals of group A (SEM ×10,000) and (b) folic acid treatedanimals in group B (36 h) revealed similar deposit of precipitated crystals in theB×
igiilttapbootfbaiwssatookt
Acknowledgement
owman’s capsule which was not visible in the animals of control group (SEM5500).
ntervals LPO levels were diminished after 36 h administration sug-esting restoration of renal injury caused by folic acid. This decreasen LPO levels after 36 h administration could be due to repletion ofntracellular GSH as observed in group B. The increased LPO levelseading to membrane damage were also supported by observing thehree-dimensional architecture of the renal glomerulus in folic acidreatment groups (A and B) by scanning electron microscopy. Acutedministration of folic acid resulted in the dramatic changes in mor-hology of podocytes. Bulging nucleated cell bodies of podocytesecame swollen in folic acid treated kidneys due to an accumulationf variable sized intracellular vacuoles and granules. Appearancef collapsed podocyte cell bodies were probably due to the rup-uring of large intracellular vacuoles. The major processes arisingrom cell bodies became flattened, less discrete and also exhibitedulbous protrusions of their surfaces suggesting membrane dam-ge which could be due to generation reactive oxygen metabolitesn renal tissue. Occasionally, pore-like openings of variable size
ere visible on the cell bodies and major processes which repre-ent sites of exocytosis of the podocyte vacuoles into the urinarypace. Cox et al. (1974) had observed that podocyte major processesnd pedicels appeared crowded and indistinguishable in responseo ARF resulting from the vasoconstriction produced by infusionf nor-epinephrine. There was significant reduction in the number
f erythrocytes in and around the glomerulus in folic acid treatedidneys. Blebbing of villi and considerable brush border degenera-ion in proximal convulated tubules following folic acid treatment
ogic Pathology 64 (2012) 225– 232 231
indicates apoptosis and severe nephrotoxicity in renal tissue whichcould be attributed to increased oxidative stress. Due to its poorsolubility, folic acid at acid pH precipitates it intratubularly as aconsequence of fluid reabsorption and increasing acidity along thenephron (Huguenin et al., 1978). This intratubular obstruction dueto folic acid crystals could be the primary cause of ARF inducedby acute administration of folic acid. The alterations in membranearchitecture following folic acid treatment viewed by SEM were fur-ther substantiated by estimating the antioxidant enzyme activities.Since lipid peroxides are the substrates for GSH-Px and are precur-sors of MDA, it is quite possible that there is a possible link betweenincreased LPO and reduced GSH-Px observed in FA-ARF. Recentstudies have demonstrated a protective function for extracellularGSH-Px within the kidney. Treatments that reduced renal extra-cellular/plasma GSH-Px protein production were shown to resultin oxidative damage in the kidney (Dobashi et al., 1999). Renalextracellular GSH-Px overproduction in transgenic mice protectedtheir kidneys from damage in ischemia/perfusion experiments asevidenced by decreased BUN levels, decreased tubular necrosisand apoptosis and decreased LPO (Ishibashi et al., 1999). GSH-Px levels were significantly decreased in the animals of group Awhere as the decrease was marginal in case of group B indicatingthat a prolonged renal insult following folic acid administrationrender decreased antioxidant enzyme protection in the kidney.Antioxidant enzymes, SOD and CAT are the first line of defenseagainst oxidative stress. SOD offers protection from highly reactivesuperoxide anions (O2
•−) and converts them to hydrogen peroxide(Halliwell, 1992). We have investigated the possibility that reducedrenal antioxidant enzyme protection may contribute to oxidativestress in FA-ARF. Acute administration of folic acid was observed toreduce SOD activity in the mice kidney, in both the groups. How-ever the reduction in SOD activity was significantly higher in groupA as compared to the other group. A continuous folic acid insultmay exacerbate the oxidative stress in the kidney. CAT is respon-sible for the catalytic decomposition of H2O2 to O2 and H2O. Thedecreased CAT activity in response to acute administration of folicacid might reduce the protection against free radicals. It is clearthat the simultaneous reduction in the activity of both SOD andCAT makes the kidney more vulnerable to folic acid induced oxida-tive stress. However we did not observe any significant change inGST activity following folic acid administration. Various authorshave reported an increased prooxidant state and reduced antiox-idant potential in experimental ARF models (Priuska and Schacht,1995; Guidet and Shah, 1989; Abdul-Ezz et al., 1991; Sugihara andGemba, 1986; Wolf et al., 1994) but to date no such report exist forFA-ARF. For the first time we have been reporting that folic acid, anantioxidant itself, in high concentration could lead to an increasedprooxidant state and decreased protection against oxidative stressby depleting the antioxidant enzyme activities.
6. Conclusion
In conclusion, we suggest that the induction of a prooxidantstate could be the key mechanism for initiation of ARF followingacute administration of folic acid. Moreover, the observations alsodemonstrated that the short-term administration of a high doseof folic acid renders an increased pathological state in the renaltissue than single acute dose of folic acid. It has been suggested thatantioxidant therapy might give helpful results in the prevention ofFA-ARF.
This work was supported by the grant sanctioned to Dr. SanjeevPuri by UGC-funded Centre with Potential for Excellence in Biomed-
2 oxicol
iai
R
A
AB
B
B
B
C
C
D
D
D
E
F
F
F
G
H
H
H
I
J
Wolf A, Clemann N, Frieauff W, Ryffel B, Cordier A. Role of reactive oxygen formation
32 A. Gupta et al. / Experimental and T
cal Science (CPEBS), Panjab University, Chandigarh. The financialssistance from the Indian Council of Medical Research, New Delhi,s gratefully acknowledged (Grant No. 45/10/2007/CMB/BMS).
eferences
bdul-Ezz SR, Walker PD, Shah SV. Role of glutathione in an animal model of myo-globinuric acute renal failure. Proc Natl Acad Sci 1991;88:9833–7.
ebi H. Catalase in vitro. Meth Enzymol 1984;105:121–6.aliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanism in toxic acute renal failure.
Drug Met Rev 1999;31(4):971–97.aserga R, Thatcher D, Marzi D. Cell proliferation in mouse kidney after single injec-
tion of folic acid. Lab Invest 1968;19:92–6.aud L, Ardaillou R. Reactive oxygen species: production and role in the kidney. Am
J Physiol 1986;251:F756–776.osch RJ, Woolf AS, Fine LG. Gene transfer into the mammalian kidney: direct
retrovirus transduction of regenerating tubular epithelial cells. Exp Nephrol1993;1:49–54.
heng CW, Rifai A, Ka SM, Shui HA, Lin YF, Lee WH, Chen A. Calcium-binding proteinsannexin A2 and S100A6 are sensors of tubular injury and recovery in acute renalfailure. Kidney Int 2005;68:2694–703.
ox JW, Baehler RW, Sharma H, O’Dorisio T, Osgood RW, Stein JH, Ferris TF. Studiesof the mechanism of oliguria in a model of unilateral acute renal failure. J ClinInvest 1974;53:1546–58.
ai C, Yang J, Liu Y. Single injection of naked plasmid encoding hepatocyte growthfactor prevents cell death and ameliorates acute renal failure in mice. J Am SocNephrol 2002;13:411–22.
obashi K, Asayama K, Nakane T, Hayashibe H, Kodera K, Uchida N, Nakazawa S.Effect of peroxisome proliferator on extracellular glutathione peroxidase in rat.Free Radic Res 1999;31:181–90.
oi K, Okamoto K, Negishi K, et al. Attenuation of folic acid-induced renal inflam-matory injury in platelet-activating factor receptor-deficient mice. Am J Pathol2006;168(5):1413–24.
sterbauer H, Schaur R, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal,malonaldehyde and related aldehydes. Free Radic Biol Res 1991;11:81–128.
ang TC, Alison MR, Cook HT, Jeffery R, Wright NA, Poulsom R. Proliferation of bonemarrow-derived cells contributes to regeneration after folic acid-induced acutetubular injury. J Am Soc Nephrol 2005;16(6):1723–32.
iaschi-Taesch NM, Santos S, Reddy V, Van Why SK, Philbrick WF, Ortega A, Esbrit P,Orloff JJ, Garcia-Ocana A. Prevention of acute ischemic renal failure by targeteddelivery of growth factors to the proximal tubule in transgenic mice: the efficacyof parathyroid hormone-related protein and hepatocyte growth factor. J Am SocNephrol 2004;15:112–25.
lohe L, Gunzler WA. Assays of glutathione peroxidase. Meth Enzymol1984;105:114–21.
uidet B, Shah SV. Enhanced in vivo hydrogen peroxide generation by rat kidney inglycerol-induced renal failure. Am J Physiol 1989;257:F440–5.
alliwell B. Reactive oxygen species and the central nervous system. J Neurochem1992;59:1609–23.
augen E, Nath K. The involvement of oxidative stress in the progression of renalinjury. Blood Purif 1999;17:58–65.
uguenin ME, Birbaumer A, Brunner FP, Thorhorst J, Schmidt U, Dubach UC, Thiel G.An evaluation of the role of tubular obstruction in folic acid induced acute renalfailure in the rat. Nephron 1978;22:41–54.
shibashi N, Weisbrot-Lefkowitz M, Reuhl K, Inouye M, Mirochnitchenko O. Modula-tion of chemokines expression during ischemia/reperfusion in transgenic miceoverproducing human glutathione peroxidases. J Immunol 1999;163:5666–77.
oannidis M, Bonn G, Pfaller W. Lipid peroxidation-An initial event in experimentalARF. Renal Physiol Biochem 1989;12:47–55.
ogic Pathology 64 (2012) 225– 232
Klinger EL, Evan AP, Anderson RE. Folic acid-induced renal injury and repair-correlation of structural and functional abnormalities. Arch Pathol Lab Med1980;104:87–93.
Kono Y. Generation of superoxide radical during autoxidation of hydroxylamine andan assay for superoxide dismutase. Arch Biochem Biophys 1978;186:189–95.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folinphenol reagent. J Biol Chem 1951;193:265–75.
Maser RL, Vassmer D, Magenheimer BS, Calvet JP. Oxidant stress and reducedantioxidant enzyme protection in polycystic kidney disease. J Am Soc Nephrol2002;13:991–9.
Metnitz GH, Fischer M, Bartens C, Steltzer H, Lang TH, Druml W. Impact of ARF onantioxidant status in multiple organ failure. Acta Anaesthesiologica Scandinav-ica 2000;44(3):236–40.
Mullin EM, Bonar RA, Paulson DF. Acute tubular necrosis: an experimental modeldetailing the biochemical events accompanying renal injury and recovery. InvestUrol 1976;13:289–94.
Nabae K, Doi Y, Takahashi S, Ichihara T, Toda C, Ueda K, Okamoto Y, Kojima N,Tamano S, Shirai T. Toxicity of di (2-ethylhexyl)phthalate (DEHP) and di (2-ethylhexyl)adipate (DEHA) under conditions of renal dysfunction induced withfolic acid in rats: Enhancement of male reproductive toxicity of DEHP is associ-ated with an increase of the mono-derivative. Reprod Toxicol 2006;22:411–7.
Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobar-bituric acid reaction. Anal Biochem 1979;95:351–8.
Ortega A, Ramila D, Izquierdo A, Gonzalez L, Barat A, Gazapo R, Bosch RJ, EsbritP. Role of the renin-angiotensin system on the parathyroid hormone-relatedprotein overexpression induced by nephrotoxic acute renal failure in the rat. JAm Soc Nephrol 2005;16:939–49.
Ortiz A, Lorz C, Catalan MP. Expression of apoptosis regulatory proteins in tubu-lar epithelium stressed in culture or following acute renal failure. Kidney Int2000;57:969–81.
Paller MS, Hoidal JR, Ferris TF. Oxygen free radicals in ischemic acute renal failurein the rat. J Clin Invest 1984;72:1156–64.
Polat A, Parlakpinar H, Tasdemir S, Colak C, Vardi N, Ucar M, Emre MH, Acet A.Protective role of aminoguanidine on gentamicin-induced acute renal failure inrats. Acta Histochemica 2006;108:365–71.
Priuska EM, Schacht J. Formation of free radicals by gentamicin and iron and evidencefor an iron/gentamicin complex. Biochem Pharmacol 1995;50:1749–52.
Roberts JC, Francetic DJ. The importance of sample preparation and storage in glu-tathione analysis. Anal Biochem 1993;211:183–7.
Sugihara K, Gemba M. Modification of cisplatin toxicity by antioxidants. Jpn J Phar-macol 1986;40:353–5.
Szczypka MS, Westover AJ, Clouthier SG, Ferrara JL, Humes HD. Rare incorporationof bone marrow-derived cells into kidney after folic acid-induced injury. StemCells 2005;23:44–54.
Threlfall G. Cell proliferation in the rat kidney induced by folic acid. Cell Proliferation1968;1:383–92.
Tsutsumi T, Ichihara T, Kawabe M, Yoshino H, Asamoto M, Suzuki S, Shirai T. Renaltoxicity induced by folic acid is associated with the enhancement of malereproductive toxicity of di (n-butyl)phthalate in rats. Reprod Toxicol 2004;18:35–42.
Wan B, Hao L, Qiu Y, Sun Z, Cao Q, Zhang Y, Zhu T, Wang H, Zhang Y. Blocking tumornecrosis factor-� inhibits folic acid-induced acute renal failure. Exp Mol Pathol2006;81:211–6.
Warholm M, Guthenberg C, Von Bahr C, Mannervik B. Glutathione transferases fromhuman liver. Meth Enzymol 1985;113:499–504.
in the cyclosporine A-mediated impairment of renal functions. Transplant Proc1994;26:2902–7.
Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its impli-cations for health. J Nutr 2004;134:489–92.
