Reversal of iron-induced nephrotoxicity in rats by
molsidomine,a nitric oxide donor
Amit Gupta,Sameer Sharma,Kanwaljit Chopra
*
Pharmacology Division,University Institute of Pharmaceutical Sciences,Panjab University,Chandigarh 160014,India
Received 18January 2007;accepted 24August 2007
Abstract
Fe-NTA is a very potent nephrotoxic agent and causes oxidative renal injury as shown in various studies.Reactive oxygen species as well as nitric oxide (NO)play an important role in acute renal failure (ARF).Present study was designed to investigate the effect of NO donor,molsidomine (Mol)and inducible NO synthase inhibitor (iNOS),aminoguanidine (AG)in Fe-NTA-induced renal toxicity.Rats were pretreated with Mol (5,7.5and 10mg/),and AG (100mg/kg,i.p.)before Fe-NTA challenge (8mg iron/kg body wei
ght,i.p.)to determine the urea and creatinine levels along with biochemical analysis of oxidative stress.Fe-NTA administration markedly increased the BUN and serum creatinine level which was coupled with a marked lipid peroxidation,reduced activity of glutathione and decreased total nitric oxide levels of rat kidneys coupled with significant morphological alterations.Fe-NTA also markedly increased the levels of tumor necrosis factor-a (TNF-a )in serum.Concomitant treatment with molsidomine significantly reduced the serum cre-atinine and BUN levels,decreased lipid peroxidation in a significant manner,restored the levels of reduced glutathione,increased total nitric oxide levels and restored the normal morphology.Molsidomine treatment also attenuated the serum levels of TNF-a .Prior admin-istration of AG did not reverse the protective effects produced by molsidomine.Present findings strongly suggest that protection afforded by molsidomine may be due to its direct NO donor ability but not through nitric oxide synthase activity as pretreatment with amino-guanidine did not abolish the protective effects of molsidomine.Ó2007Elsevier Ltd.All rights reserved.
Keywords:Acute renal failure;Nitric oxide;Nitric oxide synthase;Molsidomine;Aminoguanidine;Oxidative stress
1.Introduction
It has been demonstrated that an iron-chelate of nitri-lotriacetate,Fe-NTA induces acute and sub-acute renal injury in animals (Awai et al.,1979;Hamazaki et al.,1985).Repeated intraperitoneal administration of ferric nitrilotriacetate (Fe-NTA)was reported to cause high degree of renal adeno-carcinoma in rats and mice (Okada and Midorikawa,1982;Hamazaki et al.,1986;Okada,1996).It is reported that Fe-NTA administration can spe-cifically causes allelic loss of the p16tumor suppressor gene
in renal tubular cells (Hiroyasu et al.,2002).Some studies have reported that oxygen free radical was formed from redox-active iron and was detected in the serum of Fe-NTA-treated rats (Liu et al.,1993;Zhang et al.,1995).Excess free iron causes free radical-mediated peroxidation of membrane lipids and leads to generation of malondial-dehyde (MDA)and 4-hydroxy-2-nonenal (HNE)(Jacobs,1980;Preece et al.,1988;Toyokuni et al.,1994;Chen et al.,1998).Iron-catalyzed oxidative stress is believed to be the main culprit involved in the pathogenesis of iron-induced cancer (Toyokuni 1996,2002).It is considered that reactive oxygen species (ROS)induced by iron originate from the interaction of iron with superoxide (O 2À)gener-ated via Fenton/Haber–Weiss or auto-oxidation reaction (Beckman and Koppenol,1996;Liochev and Fridovich,2002).
0278-6915/$-see front matter Ó2007Elsevier Ltd.All rights reserved.doi:10.1016/j.fct.2007.08.041
Abbreviations:ARF,acute renal failure;AG,aminoguanidine;Fe-N-TA,ferric nitrilotriacetate;Mol,molsidomine;NO,nitric oxide;TNF-a ,tumor necrosis-alpha.*
Corresponding author.Tel.:+911722534105;fax:+911722541142.E-mail address:dr_chopra_k@yahoo (K.Chopra).www.elsevier/locate/foodchemtox
Available online at www.sciencedirect
Food and Chemical Toxicology 46(2008)
537–543
It is well known that excess free iron induces the expression of iNOS,releases the nitric oxide which com-bines with superoxide anions to form‘peroxynitrite’,a very toxic mediator of lipid peroxidation as well as oxida-tive damage to cellular membrane(Chen et al.,1998, 2001).A basal production of NO is necessary for main-taining the normal physiology of renal hemodynamics as well as adequate glomerular function(Waz et al.,1998). So,inhibition of NO may exacerbate the renal dysfunc-tion,while NO modulators may improve the renal func-tion and physiology.Iqbal et al.(2003)have shown that when exogenously NO is given,it preserves the renal architecture and prevents the oxidative damage to the cel-lular components.There are different pathway by which NO shows its antioxidant mechanisms against iron-cata-lyzed oxidative damage which have been reviewed by Kagan et al.(2001).
Molsidomine(Mol)is a prodrug,decarboxylates in liver via enzymatic action to form SIN-1(Kukovetz and Holzmann,1986).It is a potent vasodilator,so widely used for treatment of angina.It increases cyclic GMP (cGMP)by stimulating guanylate cyclase,thus causing smooth muscle relaxation(Gruetter et al.,1979).SIN-1 has been shown to have beneficial effects in reperfusion injury,reducing neutrophil sequestration and vascular resistance,as well as preserving endothelial dysfunction (Clark et al.,1999).
Whether molsidomine mediates its protective effects via modulating the activity of inducible nitric oxide synthase(iNOS);aminoguanidine,a potent inhibitor of iNOS was used to explore this effect(Sahna et al.,2006).
In order to study the involvement of nitric oxide in renal damage,the present study was designed to investigate the effect of molsidomine and iNOS inhibitor,aminoguanidine in Fe-NTA-induced oxidative damage in the rat kidney.
2.Materials and methods
2.1.Animals
Male Wistar rats(150–200g)bred in the central animal house of Panjab University(Chandigarh,India)were used.The animals were housed under standard conditions of light and dark cycle with free access to food and water.The experimental protocols were approved by the institutional ethical committee of Panjab University,Chandigarh.
2.2.Drugs
Molsidomine(Mol)was purchased from Cayman Chemicals,USA. Disodium nitrilotriacetate(Na2NTA)an
d aminoguanidine(AG)were purchased from Sigma(St.Louis,MO,USA)and Ferric nitrate was purchased from E.Merck(India)Ltd.(Mumbai,India).
2.3.Preparation of ferric nitrilotriacetate(Fe-NTA)solution
Preparation of Fe-NTA solution was done by method given by Awai et al.(1979).Briefly,ferric nitrate(0.16mM)solution was mixed with a4-fold molar excess of disodium salt of NTA(0.64nM)and the pH was adjusted to7.4with a sodium bicarbonate solution.The solution was prepared immediately before each protocol.2.4.Preliminary studies
Molsidomine was suspended in0.25%sodium carboxymethyl cellulose and was given orally30min before Fe-NTA insult.During the pilot studies,dose of5,7.5and10mg/kg were tested and the dose of10mg was found to be most effective in the prevention of renal dysfunction and further study was carried using this dose.
2.5.Study design
Five groups were employed in the present study,each comprising of five to six animals.Group I comprised the control group that received water for injection(vehicle for Fe-NTA and Mol)only.Group II
animals received Fe-NTA(8mg iron/kg body weight,i.p.).Group III animals received AG(100mg/kg,i.p.)30min prior to the administration of Fe-NTA.Group IV animals received Mol(10mg/kg,p.o,respectively) 30min before the administration of Fe-NTA.Group V animals received AG(100mg/kg,i.p.)30min before the administration of Mol followed by Fe-NTA challenge.
Dose of aminoguanidine was selected based on the previous studies performed in our laboratory(Chander and Chopra,2005b).Four hours after Fe-NTA administration,animals were sacrificed with a high dose of anesthesia and the blood was collected in centrifuge tubes through abdominal aorta.The blood samples were centrifuged and plasma was collected.Both kidneys were harvested through a midline incision,right kidney was deep frozen till further enzymatic estimation,whereas left kidney was stored in10%formalin for the histological sectioning.
2.6.Assessment of renal function
Serum samples were assayed for blood urea nitrogen(BUN)and serum creatinine by using standard diagnostic kits(Span Diagnostics,Gujarat, India).
2.7.Post mitochondrial supernatant preparation(PMS)
After sacrificing the animals,their kidneys were quickly removed,per-fused immediately with ice-cold normal saline and homogenized in chilled potassium chloride(1.19%)using a Potter Elvenhjem homogenizer.The homogenate was differentially centrifuged to obtain the post mitochondrial supernatant(PMS),which was used for further enzymatic analysis.
2.8.Estimation of lipid peroxidation
The malondialdehyde(MDA)content,a measure of lipid peroxida-tion,was assayed in the form of thiobarbituric acid reacting substances (TBARS)(Ohkawa et al.,1979).In brief,the reaction mixture consisted of 0.2ml of8.1%sodium lauryl sulfate,1.5ml of20%acetic acid solution adjusted to pH3.5with sodium hydroxide and1.5ml of0.8%aqueous solution of thiobarbituric acid was added to0.2ml of10%(w/v)of PMS. The mixture was brought up to4.0ml with distilled water and heated at 95°C for1h.After cooling with tap water,1.0ml of distilled water and 5.0ml of the mixture of n-butanol and pyridine(15:1v/v)was added, shaken well and centrifuged.The organic layer was taken out and its absorbance was measured at532nm.TBARS were quantified using extinction coefficient of1.56·105MÀ1cmÀ1and expressed as nmol of TBARS per mg protein.Tissue protein was estimated using the Biuret method of protein assay and the renal MDA content expressed as nano-moles of malondialdehyde per milligram of protein(Varley,1988).
2.9.Estimation of renal antioxidant pool
The reduced glutathione(GSH)was measured by the method of Jollow et al.(Jollow et al.,1974).The yellow colour developed by the reduction of Ellman’s reagent by–SH groups of GSH was read at412nm.The superoxide dismutase(SOD)activity was assessed by the method of Kono
538 A.Gupta et al./Food and Chemical Toxicology46(2008)537–543
(1978)were the auto-oxidation of hydroxylamine in presence of PMS was observed by measuring the absorbance at560nm.
reactive materials studies2.10.Estimation of renal nitrite levels
Nitrite levels were estimated using Griess reagent which served as an indicator of nitric oxide production(Di Rosa et al.,1990).Briefly,1.0ml of Griess reagent(1:1solution of1%sulphanilamide in5%phosphoric acid and0.1%napthylethylene diamine dihydrochloride in water)was added to1.0ml of PMS and absorbance was measured at546nm nitrite concentration was calculated using a standard curve for sodium nitrite and nitrite levels were expressed as l mol/mg protein.
2.11.Estimation of tumor necrosis factor-alpha
Tumor necrosis factor-alpha(TNF-a)was estimated using a standard ELISA method in serum of the rats.The estimations were done by sandwich ELISA method according to manufacturer’s instructions(R&D Systems).
2.12.Renal histology
The left kidney was isolated immediately after sacrificing the animal and washed with ice-cold saline.It was thenfixed in a10%neutral buffered formalin solution,embedded in paraffin and used for histological examina-tion.Five-micrometer(l m)thick sections were cut,deparaffinized,hydrated and stained with hematoxylin and eosin.The renal sections were examined in blind fashion for hemorrhagic and hyaline casts,tubular necrosis and apical blebbing in all treatments.A minimum of10fields for each kidney slide were examined and assigned for the severity of changes using scores on a scale of none(À),mild(+),moderate(++)and severe(+++)damage.
2.1
3.Statistical analysis
Results are expressed as mean±SEM.One-way analysis of variance (ANOVA)followed by Dunnett’s te
st was applied to calculate the statis-tical significance between various groups.A value of p<0.05was con-sidered to be statistically significant.
3.Results
3.1.Effect of molsidomine on Fe-NTA-induced renal dysfunction
Fe-NTA administration resulted in a significant increase in serum creatinine and blood urea nitrogen(BUN)levels as compared to control animals.Pretreatment with Mol (10mg/)30min before Fe-NTA administration markedly improved renal dysfunction(indicated by reduced serum creatinine and BUN levels).The effect was found to be in a dose-dependent manner during the preli-minary studies.(Fig.1a and b).
3.2.Effect of molsidomine oxidative stress markers in control and Fe-NTA-treated rats
3.2.1.Effect of molsidomine on renal MDA and GSH levels
Fe-NTA-treated rats showed a significant lipid peroxi-dation as indicated by a marked increase in the renal TBARS levels and decreased GSH levels as compared to vehicle-treated control rats.Pretreatment with a higher dose of Mol(10mg/)significantly reduced the TABRS levels and r
estored the GSH levels as compared to Fe-NTA-treated group.AG treatment produced no effect on TBARS or GSH levels as compared to Mol trea-ted animals(Fig.2a and b).
3.2.2.Effect of molsidomine on renal SOD and nitrite levels
Treatment with Fe-NTA significantly decreased the enzymatic activity of renal superoxide dismutase (SOD)and nitrite levels.Administration of molsido-mine significantly reversed the decrease in renal SOD and nitrite levels as compared to Fe-NTA-treated rats. However,the protection afforded by molsidomine was not abolished by treatment with aminoguanidine (Fig.2c and d).
3.3.Effect of molsidomine on serum tumor necrosis
factor-a(TNF-a)levels
Fe-NTA administration significantly increased the serum TNF-a level as compared to control group.Pretreat-ment with molsidomine significantly attenuated the levels serum TNF-a.AG treatment also decreased the serum levels of TNF-a(Table2).
A.Gupta et al./Food and Chemical Toxicology46(2008)537–543539
3.4.Effect of molsidomine on Fe-NTA-induced changes in renal morphology
The histopathological changes were graded and summa-rized in Table1.The control group did not show any mor-phological changes.By contrast,the kidneys of rats treated with Fe-NTA showed marked histological changes in cor-tex and outer medulla.The renal sections showed severe epical blebbing,hyaline casts and tubular necrosis.Molsi-domine treated kidney sections preserved the normal mor-phology of the kidney.Aminoguanidine treatment in Mol+Fe-NTA-treated group showed si
milar morphology as that of Fe-NTA-treated animals(Fig.3).
4.Discussion
Iron-induced toxicity has been observed in-vivo in whole kidney,in freshly isolated as well as cultured proximal tubule segments(Chen et al.,1998;Wu and Qiu,2001; Kadkhodaee and Gol,2004).To elucidate the role of NO as a factor contributing to iron-mediated injury,the effects of modulation of NO levels by molsidomine,a NO direct donor and aminoguanidine,a specific iNOS inhibitor,on Fe-NTA-induced nephrotoxicity were evaluated.
Fe-NTA has shown to cause acute as well as sub-acute nephrotoxicity in various studies(Shimoi et al.,1997;Egu-chi et al.,1999;Wu and Qiu,2001).The reduction Fe3+-NTA to Fe2+-NTA results in the generation of superoxide radicals(O2À)which potentiate the iron-catalyzed Haber–Weiss reaction to produce hydroxyl radical(OH),resulting
Table1
Effect of molsidomine(10mg/kg)and aminoguanidine(100mg/kg)on morphological changes as assessed by histopathological examination of kidney of Fe-NTA-treated rats
Groups Tubular
brush
border loss Tubular
necrosis
Hyaline
casts
Epical
blebbing
Group1ÀÀÀÀ
Group2+++++++++++
Group3+++++++++++
Group4À+/À+/ÀÀ
Group5++++
Key:(À)absent;(+/À)variable;(+)very less;(++)moderate;and(+++)
severe.
540 A.Gupta et al./Food and Chemical Toxicology46(2008)537–543
in the progression of lipid peroxidation and oxidative DNA damage(Aruoma et al.,1989;Umemura et al., 1990b).
In the present study,single intraperitoneal injection of Fe-NTA(8mg iron per kg body weight)impaired the renal function leading to marked increase in serum creatinine and BUN;significantly increased the renal TBARS levels and depleted the antioxidant enzyme pool as it is evident from the decreased level
s of reduced glutathione and SOD.Administration of Fe-NTA significantly reduced the renal NO levels and markedly increased the serum TNF-a levels.Moreover,the kidney of Fe-NTA-treated rats showed characteristic morphological changes such as tubular brush border loss,tubular necrosis and hyaline casts.Pretreatment with molsidomine significantly improved the impaired renal function,reduced renal TBARS,and increased the depleted renal antioxi-dant enzymes(GSH and SOD)as well as renal nitrite levels.Also,administration of molsidomine significantly
Table2
Effects of molsidomine(10mg/)and aminoguanidine(100mg/kg,
i.p.)on serum tumor necrosis factor-alpha(TNF-a)levels
Drug treatment Serum TNF-a levels(pg/ml)
Control32.33±1.43
Fe-NTA153.33±2.37*
Mol+Fe-NTA95.26±2.12**
AMG+Mol+Fe-NTA127.41±1.58***
The values are expressed as mean±S.E.M.
*p<0.05as compared to control.
**p<0.05as compared to Fe-NTA-treated group.
***p<0.05as compared to drug treated groups(one-way ANOVA fol-
lowed by Dunnett’s
test).
Fig.3.Hemotoxylin and eosin stained sections of rat kidney:(a)Normal kidney section.(b)Kidney section of Fe-NTA-treated rat showing tubular damage,hyaline cast formation and brush border loss.(c)Kidney section of AG+Fe-NTA-treated rat showing morphological damage similar to Fe-NTA-treated rats.(d)Kidney section of Mol10+Fe-NTA-treated rat showing a near normal morphology.(e)Kidney section of AG+Mol10+Fe-NTA-treated rat showing a near normal morphology.
A.Gupta et al./Food and Chemical Toxicology46(2008)537–543541
improved the deteriorated renal architecture.Prior treat-ment of animals with AG(100mg/kg,i.p.)did not abolish the protective effects of molsidomine in Fe-NTA-treated rats.
Earlier studies have demonstrated the critical role of iron in the formation of ROS that ultimately cause perox-idative damage to vital cell structures(Toyokuni,2002).It may be quite possible that excessive production of superox-ide radicals due to iron administration(via Fenton’s reac-tion chemistry)reacts with renal NO,generated via increased iNOS expression,thus leading to the formation of peroxynitrite,which itself along with superoxide radicals result in loss of renal function as well as oxidative damage. NO plays a very crucial role in maintaining the normal physiology of renal hemodyn
amics and glomerular func-tion(Waz et al.,1998).It has been shown that non-heme free iron is responsible for the activity of NOS through its control on nuclear transcription of NOS mRNA.Fur-thermore,altered iron stores causes inflammatory condi-tions characterized by increased macrophage activity and enhanced cytokine levels(Weinberg,1992).Oxidative stress is also one of the major stimulators for the formation reac-tive nitrogen species(RNI)which causes oxidative and tis-sue damage,resulting in the activation of NF-r B which ultimately results in the elevated levels of inflammatory cytokines(Unanue and Dixon,1967).
Nitric oxide also results in the activation of heme-oxy-genase-1which affords protection from the iron-induced renal injury(Aggarwal and Nick,2000).Many workers have studied the protective role of exogenous administra-tion of NO in various models of acute as well as chronic renal failure(Clancy et al.,1992;Chintala et al.,1993; Iqbal et al.,2003).Kagan et al.(2001)have proposed the antioxidant mechanisms of NO which prevents iron-cata-lyzed cellular oxidative damage.
Molsidomine has been previously reported to have pro-tective effects in various renal complications as well as iron-induced renal injury(Rodriguez-Pena et al.,2004;Chander and Chopra,2005a,c).Our study also demonstrated that by augmenting the NO levels,molsidomine protected the renal injury and this protection was not reversed by treat-ment by aminoguanidine,thus clearly indicating the mols-idomine
produces its effect by direct nitric oxide donation property and does not involve modulation of NOS in ame-liorating iron-induced renal injury.
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