Effect of molsidomine and l -arginine in cyclosporine nephrotoxicity: role of nitric oxide

Toxicology 207 (2005) 463–474

Effect of molsidomine and l-arginine in cyclosporine nephrotoxicity: role of nitric oxide Vikas Chander, Kanwaljit Chopra∗ Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India Received 24 September 2004; received in revised form 26 October 2004; accepted 27 October 2004 Available online 21 December 2004

Abstract Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. To determine if the renal alterations are mediated directly by cyclosporine or by secondary homodynamic alterations induced by cyclosporine, we evaluated if l-arginine and a nitric oxide donor, molsidomine could prevent these alterations. Eight groups of rats were employed in this study, group 1 served as control, group 2 rats were treated with CsA (20 mg/kg, s.c. for 21 days), group 3 received CsA along with l-arginine (125 mg/kg in drinking water concurrently with CsA), groups 4 and 5 received CsA along with molsidomine (5 and 10 mg/kg, p.o. 24 h before and 21 days concurrently with CsA), group 6 received CsA along with l-arginine (125 mg/l in drinking water concurrently with CsA) and l-NAME (10 mg/kg), groups 7 and 8 received l-NAME (10 mg/kg) along with CsA and molsidomine (5 and 10 mg/kg), respectively. Renal function was assessed by measuring serum creatinine, blood urea nitrogen, creatinine and urea clearance. Tissue and urine nitrite and nitrate levels were measured to estimate the total nitric oxide levels. The renal oxidative stress was measured by renal malondialdehyde levels, reduced glutathione levels and by enzymatic activity of catalase and superoxide dismutase. Renal morphological alterations were assessed by histopathological examination. CsA administration for 21 days resulted in a marked renal oxidative stress, significantly deranged the renal functions as well as renal morphology. Treatment with l-arginine as well as with molsidomine significantly improved the renal dysfunction; tissue and urine total nitric oxide levels, renal oxidative stress and prevented the alterations in renal morphology. This protection against CsA nephrotoxicity was attenuated by treatment with l-NAME, clearly indicating that NO plays a pivotal role in renoprotective effect of l-arginine and molsidomine against cyclosporine nephrotoxicity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Cyclosporine A (CsA); Nephrotoxicity; Nitric oxide (NO); l-Arginine; Molsidomine; l-NAME

1. Introduction ∗

Corresponding author. Tel.: +91 172 2534105; fax: +91 172 2541142. E-mail address: dr chopra [email protected] (K. Chopra).

Cyclosporine (CsA) is an immunosuppressive drug widely used in the management of all transplantation and to treat autoimmune diseases. The clinical usage of

0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.10.018

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CsA is however restricted due to both nephrotoxicity and hypertension (Busauschina et al., 2004). The renal damage consequent to CsA administration ranges from hemodynamic alterations to irreversible chronic lesions. CsA nephrotoxicity is characterized by renal vasoconstriction that induces a decrease in renal plasma flow and glomerular filtration rate. This side effect is usually reversible when CsA dose is reduced. However, even with normal blood levels or during longterm dosage, CsA can produce chronic hypertension and can be the cause of irreversible impairment of renal function (Shihab et al., 2000). Renal vasoconstriction is attributed to an imbalance in the release of vasoactive substances: on one hand, increased release of vasoconstrictive factors such as thromboxane (Perico et al., 1986b), endothelin (Kon et al., 1990), and angiotensin II (Perico et al., 1986a); and on the other, a decrease in vasodilating factors such as prostacyclin (Perico et al., 1986a) and nitric oxide (NO) (Vaziri et al., 1998; Gossmann et al., 2001). Acute CsA nephrotoxicity may appear soon after transplantation or after weeks or months, with oliguria, acute decrement of glomerular filtration rate and renal plasma flow (Kahan, 1989). Due to lack of histological lesions, the acute toxicity is usually believed to be consequent to renal vasoconstriction (Kahan, 1989). Conversely, after prolonged CsA administration, chronic nephrotoxicity is characterized by a progressive and mostly irreversible impairment of renal function, and it is supported by histological lesions ranging from striped fibrosis to ischemic collapse of the tuft, glomerular sclerosis and tubular atrophy (Amore et al., 1995), however, the increase in interstitial matrix preceding the interstitial fibrosis might be due to a direct toxic effect of CsA (Amore et al., 1995). NO is a vasoactive factor that plays a key role in maintaining vascular tone in the kidney. It is produced from l-arginine (l-arg) by nitric oxide synthase (NOS), of which at least three molecular-level isoforms have been identified and all of these three NOS isoforms are present in the kidney. In renal cortex, nNOS exhibits a macula densa cell-specific expression, iNOS has been observed in mesangial and proximal tubule cells of the afferent and efferent arterioles and glomerular capillaries. The protective effect of l-arg had been previously addressed in nephrotoxicity induced by cisplatin (Li et al., 1994). In addition, few reports showed that admin-

istration of l-arg greatly ameliorated kidney dysfunction induced by CsA in rats (Amore et al., 1995; Yang et al., 1998). However, this has been recently disputed by Lassila et al. (2001) who showed that treatment with l-arg did not affect renal dysfunction-induced by CsA. Molsidomine a prodrug (belonging to the class of sydnonimines) is a potent vasodilator and has been used widely as an antianginal agent. In the liver, it decarboxylates enzymatically to form SIN-1 (Kukovetz and Holzmann, 1986), which does not require enzymatic bioactivation and NO is released spontaneously (Bohn and Schonafinger, 1989; Hinz and Schroder, 1999). Molsidomine and other NO-yielding compounds relax vascular smooth muscle by stimulating guanylate cyclase and thereby increasing cyclic GMP (cGMP) levels (Gruetter et al., 1979). The present work was undertaken to investigate the possible protective effect of l-arginine and molsidomine on nephrotoxicity induced by CsA.

2. Materials and methods 2.1. Chemicals Cyclosporine in powder form was gifted by Panacea Biotech Ltd. (Lalru, India), molsidomine (Caymen chemicals, USA), l-NAME (Sigma, USA), l-arginine (HiMedia, Mumbai, India). All the remaining chemicals were of highest grade commercially available.

2.2. Animals Male Wistar rats (150–200 g), 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 (Hindustan Lever Products, Kolkata, India) and water. The experimental protocols were approved by the institutional ethical committee of Panjab University, Chandigarh.

2.3. Experimental groups Animals were distributed into eight groups, each comprising of six to eight animals:

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(1) A control group received an equivalent volume of vehicle for CsA, i.e. olive oil, subcutaneously (s.c.) and saline perorally (p.o.) for 21 days. (2) The second group received CsA (20 mg/kg, s.c.) dissolved in olive oil for 21 days. (3) The rats received l-arginine (125 mg/kg in drinking water) 24 h before administering CsA, and continued along with CsA for 21 days. (4) The rats received molsidomine (5 mg/kg, p.o.) 24 h before administering CsA, and continued along with CsA for 21 days. (5) The rats received a dose of molsidomine (10 mg/kg, p.o.) 24 h before administering CsA, and continued alongwith CsA for 21 days. (6) The rats received a dose of N(G)-nitro-larginine methyl ester (l-NAME) (10 mg/kg) along with l-arginine 24 h before administering CsA, and continued along with CsA for 21 days. (7) The rats received a dose of l-NAME (10 mg/kg) and molsidomine (5 mg/kg, p.o.) 24 h before administering CsA, and continued along with CsA for 21 days. (8) The rats received a dose of l-NAME (10 mg/kg) and molsidomine (10 mg/kg, p.o.) 24 h before administering CsA, and continued along with CsA for 21 days. Because of the instability of molsidomine in solution when exposed to light, the drug solution was covered with aluminium foil. Body weights of the animals were measured every day. Systolic blood pressure (SBP) was measured from the tail of the animals using a blood pressure recorder (UGO Basile, Italy) on days 0 and 22 just before sacrificing the animals. The animals were placed in individual metabolic cages for 24 hr after the last dose for urine collection. On day 22, animals were anesthetized with thiopentone sodium (40 mg/kg, i.p.) and blood was collected in heparinized centrifuge tubes through abdominal aorta. The blood samples were centrifuged and plasma was collected. A midline abdominal incision was performed and both the kidneys were isolated, the left kidney was deep frozen till further enzymatic analysis, whereas, the right kidney was stored in 10% formalin for the histological studies.

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2.4. Assessment of renal function Plasma samples were assayed for blood urea nitrogen (BUN), urea clearance, plasma creatinine and creatinine clearance by using standard diagnostic kits (Span Diagnostics, Gujarat, India). 2.5. Estimation of tissue and urine nitrite and nitrate levels Nitrite and nitrate are the primary oxidation products of NO subsequent to reaction with oxygen and therefore, the nitrite–nitrate concentration in tissue homogenate and urine was used as indicator of NO synthesis. Quantitation of nitrate and nitrite was based on the Griess reaction, in which a chromophore with a strong absorbance at 550 nm is formed by reaction of nitrite with a mixture of naphthylethylenediamine and sulphanilamide. The nitrate was reduced to nitrite by 30 min incubation with nitrate reductase in the presence of nicotinamide adenine dinucleotide 3-phosphate (NADPH). Total nitrite–nitrate concentration was calculated by using standard of sodium nitrate. Results were expressed as ␮mol/L. 2.6. Post mitochondrial supernatant preparation (PMS) After sacrificing the animals, their kidneys were quickly removed, perfused immediately with icecold normal saline and homogenized in chilled potassium chloride (1.17%) using a Potter Elvehjem homogenizer. The homogenate was differentially centrifuged to obtain post mitochondrial supernatant (PMS), which was used for further enzymatic analysis. 2.7. Estimation of renal lipid peroxides The malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid reacting substances (TBARS) (Ohkawa et al., 1979). In brief, the reaction mixture consisted of 0.2 ml of 8.1% sodium lauryl sulphate, 1.5 ml of 20% acetic acid solution adjusted to pH 3.5 with sodium hydroxide and 1.5 ml of 0.8% aqueous solution of thiobarbituric acid was added to 0.2 ml of 10% (w/v) of post mitochondrial supernatant (PMS). The mixture

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Table 1 Effect of l-arginine (125 mg/kg), molsidomine (5 and 10 mg/kg), l-NAME (10 mg/kg) treatment on body weight and SBP in chronic CsA (20 mg/kg) treated rats Treatment

Change in body weight (%)

Change in blood pressure (mm Hg) Initial (day 0)

Vehicle (olive oil) CsA (20) CsA + l-arg CsA + mol (5) CsA + mol (10) CsA + l-arg + l-NAME (10) CsA + mol (5) + l-NAME (10) CsA + mol (10) + l-NAME (10) CsA + l-NAME

0.56 −5.89 3.97 2.23 4.56 −3.56 −2.57 −3.52 −7.52

± ± ± ± ± ± ± ± ±

0.19 1.28* 0.08** 0.09** 0.89** 0.69** 0.58** 0.92** 1.86**

142 140 135 135 130 140 130 140 135

± ± ± ± ± ± ± ± ±

Final (day 22)

4.2 3.5 4.5 4.9 2.7 3.9 3.3 5.0 6.1

144 158 139 140 132 155 148 152 165

± ± ± ± ± ± ± ± ±

3.9 3.78* 5.1** 4.7** 3.4** 4.1** 5.8** 5.1** 4.8**

All values expressed as mean ± S.E.M., CsA: cyclosporine A; CsA + l-arg: l-arginine + cyclosporine A; CsA + mol (5): molsidomine (5 mg/kg) + cyclosporine A; CsA + mol (10): molsidomine (10 mg/kg) + cyclosporine A; CsA + l-arg + l-NAME (10): l-arginine + l-NAME (10 mg/kg) + cyclosporine A; CsA + mol (5) + l-NAME (10): molsidomine (5 mg/kg) + cyclosporine A + l-NAME (10 mg/kg); CsA + mol (10): molsidomine(10 mg/kg) + cyclosporine A + l-NAME (10 mg/kg); CsA + l-NAME: l-NAME (10 mg/kg) + cyclosporine A. ∗ P < 0.05 as compared to day 0 and control group. ∗∗ P < 0.05 as compared to day 0 and CsA group (one-way ANOVA followed by Dunnet’s t-test).

was brought up to 4.0 ml with distilled water and heated at 95 ◦ C for 60 min. After cooling with tap water, 1.0 ml distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15:1 v/v) was added and centrifuged. The organic layer was taken out and its absorbance was measured at 532 nm. TBARS were quantified using an extinction coefficient of 1.56 × 105 M/cm and expressed as nmol of TBARS per mg protein. Tissue protein was estimated using Biuret method (Varley, 1988)

of protein assay and the renal MDA content expressed as nanomoles of malondialdehyde per milligram of protein. 2.8. Assessment of renal antioxidant enzymes The kidney homogenate was used to assay reduced glutathione (GSH), catalase, and superoxide dismutase (SOD) activity.

Table 2 Effect of l-arginine (125 mg/kg), molsidomine (5 and 10 mg/kg), l-NAME (10 mg/kg) treatment on renal dysfunction in chronic CsA (20 mg/kg) treated rats Treatment

Plasma creatinine (mg/dl)

Vehicle (olive oil) CsA (20) CsA + l-arg CsA + mol (5) CsA + mol (10) CsA + l-arg + l-NAME (10) CsA + mol (5) + l-NAME (10) CsA + mol (10) + l-NAME (10) CsA + l-NAME

0.558 1.914 0.618 0.866 0.576 1.634 1.824 1.413 2.128

± ± ± ± ± ± ± ± ±

0.06 0.062* 0.053** 0.025** 0.03** 0.03*a 0.07*a 0.042*a 0.086*a

BUN (mg/dl) 13.86 29.45 15.79 18.55 13.95 27.12 29.06 25.72 31.05

± ± ± ± ± ± ± ± ±

0.5 0.62* 0.9** 0.6** 1.08** 0.52*a 0.46*a 0.64*a 0.58*a

Creatinine clearance (ml/min) 0.58 0.28 0.47 0.49 0.51 0.31 0.30 0.33 0.24

± ± ± ± ± ± ± ± ±

0.02 0.01* 0.005** 0.018** 0.027** 0.01*a 0.01*a 0.013*a 0.012*a

Urea clearance (ml/min) 0.608 0.228 0.572 0.584 0.586 0.276 0.276 0.228 0.2

± ± ± ± ± ± ± ± ±

0.017 0.01* 0.012** 0.02** 0.01** 0.02*a 0.01*a 0.01*a 0.01*a

All values expressed as mean ± S.E.M., CsA: cyclosporine A; CsA + l-arg: l-arginine + cyclosporine A; CsA + mol (5): molsidomine (5 mg/kg) + cyclosporine A; CsA + mol (10): molsidomine (10 mg/kg) + cyclosporine A; CsA + l-arg + l-NAME (10): l-arginine + l-NAME (10 mg/kg) + cyclosporine A; CsA + mol (5) + l-NAME (10): molsidomine (5 mg/kg) + cyclosporine A + l-NAME (10 mg/kg); CsA + mol (10): molsidomine (10 mg/kg) + cyclosporine A + l-NAME (10 mg/kg); CsA + l-NAME: l-NAME (10 mg/kg) + cyclosporine A. ∗ P < 0.05 as compared to control group. ∗∗ P < 0.05 as compared to CsA group. ∗a P < 0.05 as compared to CsA + l-arg and CsA + mol groups (one-way ANOVA followed by Dunnet’s t-test).

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2.9. Estimation of reduced glutathione Reduced glutathione in the kidney was assayed by the method of Jollow et al. (1974). Briefly 1.0 ml of PMS (10%) was precipitated with 1.0 ml of sulphosalicylic acid (4%). The samples were kept at 4 ◦ C for at least 1 h and then subjected to centrifugation at 1200 × g for 15 min at 4 ◦ C. The assay mixture contained 0.1 ml filtered aliquot and 2.7 ml

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phosphate buffer (0.1 M, pH 7.4) in a total volume of 3.0 ml. The yellow colour developed was read immediately at 412 nm on a spectrophotometer. 2.10. Estimation of catalase Catalase activity was assayed by the method of Claiborne (1985). Briefly, the assay mixture consisted

Fig. 1. Effect of l-arginine (125 mg/kg), molsidomine (5 and 10 mg/kg) and l-NAME (10 mg/kg) on CsA-induced urine and tissue nitrite levels (A and B). Values expressed as mean ± S.E.M. * P < 0.05 as compared to control group (olive oil). ** P < 0.05 as compared to CsA group. *a P < 0.05 as compared to CsA + l-arg and CsA + mol groups (one-way ANOVA followed by Dunnett’s test).

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Fig. 2. Effect of l-arginine (125 mg/kg), molsidomine (5 and 10 mg/kg) and l-NAME (10 mg/kg) on CsA-induced lipid peroxidation (MDA). Values expressed as mean ± S.E.M. * P < 0.05 as compared to control group (olive oil). ** P < 0.05 as compared to CsA group. *a P < 0.05 as compared to CsA + l-arg and CsA + mol groups (one-way ANOVA followed by Dunnett’s test).

of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml PMS (10%) in a final volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated in terms of K/min.

tro blue tetrazolium (NBT). In the cuvette 2 ml of above mixture, 0.05 ml hydroxylamine and 0.05 ml of PMS were taken and the auto-oxidation of hydroxylamine was observed by measuring the absorbance at 560 nm.

2.11. Estimation of SOD

2.12. Renal histology

SOD activity was assayed by the method of Kono (1978). The assay system consisted of EDTA 0.1 mM, sodium carbonate 50 and 96 mM of ni-

The right kidney was isolated immediately after sacrificing the animal and washed with ice-cold saline. It was then fixed in a 10% neutral buffered formalin

Table 3 Effect of l-arginine (125 mg/kg), molsidomine (5 and 10 mg/kg), l-NAME (10 mg/kg) treatment on morphological changes as assessed by histopathological examination of kidney in CsA-treated rats Group

Epical blebbing

Hyaline casts

Arteriolopathy

Tubulo interstitial fibrosis

Glomerular basement membrane thickening

C CsA (20) CsA + l-arg CsA + mol (5) CsA + mol (10) CsA + l-arg + l-NAME (10) CsA + mol (5) + l-NAME (10) CsA + mol (10) + l-NAME (10) CsA + l-NAME

− +++ + + − ++ +++ ++ +++

− +++ − − − ++ +++ ++ +++

− +++ + + − ++ +++ + +++

− ++ − − − +++ ++ ++ ++

− ++ − + − ++ + ++ +++

CsA: cyclosporine A; CsA + l-arg: l-arginine + cyclosporine A; CsA + mol (5): molsidomine (5 mg/kg) + cyclosporine A; CsA + mol (10): molsidomine (10 mg/kg) + cyclosporine A; CsA + l-arg + l-NAME (10): l-arginine + l-NAME (10 mg/kg) + cyclosporine A; CsA + mol (5) + lNAME (10): molsidomine(5 mg/kg) + cyclosporine A + l-NAME (10 mg/kg); CsA + mol (10): molsidomine (10 mg/kg) + cyclosporine A + lNAME (10 mg/kg); CsA + l-NAME: l-NAME (10 mg/kg) + cyclosporine A.

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solution, embedded in paraffin and used for histopathological examination. Five-micrometer thick sections were cut, deparaffinized, hydrated and stained with hematoxylin and eosin. The renal sections were examined in blind fashion for haemorrhagic and hyaline casts, tubulo interstitial fibrosis, arteriolopathy epical blebbing and glomerular basement thickening in all treatments. A minimum of 10 fields for each kidney slide were examined and assigned for severity of changes using scores on a scale of none (−), mild (+), moderate (++) and severe (+++) damage, in which −: denotes no abnormalities; +: changes affecting <25% of the sample; ++: changes affecting 25–50% of the sample; +++: changes affecting >50% of the sample.

3. Statistical analysis The data were analysed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test for comparing means from different treatment groups. The data was expressed as mean ± S.E.M. and a value of P < 0.05 was considered statistically significant.

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4. Results 4.1. Effect of l-arginine, molsidomine and l-NAME treatment on body weight and SBP in CsA-treated rats Chronic CsA-treated (20 mg/ml, s.c., 21 days) rats lost the body weight as compared to those receiving vehicle with the difference achieving statistical significance. This decrease in body weight was significantly improved by treatment with l-arg and molsidomine, however, this improvement in bodyweight was reversed by treatment with l-NAME. CsA administration per se caused a significant rise in SBP. The animals receiving l-arg as well as molsidomine (10 mg/kg) along with CsA reduced this rise in SBP, while this effect was reversed by treatment with lNAME (Table 1). 4.2. Effect of l-arginine, molsidomine and l-NAME treatment on CsA-induced renal dysfunction Table 2 demonstrates the effect of l-arginine, molsidomine and l-NAME on renal dysfunction

Fig. 3. Effect of l-arginine (125 mg/kg), molsidomine (5 and 10 mg/kg) and l-NAME (10 mg/kg) on CsA-induced alterations in renal antioxidant enzymes (reduced glutathione, SOD, catalase) (A–C). Values expressed as mean ± S.E.M. * P < 0.05 as compared to control group (olive oil). ** P < 0.05 as compared to CsA group. *a P < 0.05 as compared to CsA + l-arg and CsA + mol groups (one-way ANOVA followed by Dunnett’s test).

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Fig. 3. (Continued ).

induced by CSA (20 mg/(kg day−1 ) s.c. for 21 days). CsA per se administration caused an abnormal renal function in all rats. Serum creatinine and BUN were significantly increased in comparison to con-

trol rats. Pre-treatment of animals with l-arg as well as with molsidomine reduced the rise in the levels of serum creatinine and BUN, while l-NAME treatment along with these agents reversed this re-

Fig. 4. Hematoxylin and eosin stained sections of rat kidneys: (A) renal cortex of rat treated with vehicle (olive oil), (B) arteriole with marked luminal narrowing and pronounced intimal thickening in CsA rats, (C) renal cortex of rats treated with l-arg (125 mg/kg) showing no interstitial fibrosis and some arteriolopathy, (D) renal cortex of rats treated with molsidomine (5 mg/kg) showing mild interstitial fibrosis and some arteriolopathy, (E) renal cortex of rats treated with molsidomine (10 mg/kg) that prevented the development of CsA-induced alterations, (F) renal cortex of rats treated with l-arg (125 mg/kg) and l-NAME (10 mg/kg) along with CsA, showing interstitial fibrosis and arteriolopathy similar to CsA nephropathy, (G) renal cortex of rats treated with molsidomine (5 mg/kg) and l-NAME (10 mg/kg) along with CsA, showing interstitial fibrosis and arteriolopathy similar to CsA nephropathy and (H) renal cortex of rats treated with molsidomine (10 mg/kg) and l-NAME (10 mg/kg) along with CsA, showing moderate interstitial fibrosis and arteriolopathy.

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duction in renal dysfunction. There was a significant decrease in creatinine and urea clearance in CsA-treated rats, which was markedly improved by concomitant treatment with l-arginine and molsidomine. 4.3. Effect of l-arginine, molsidomine and l-NAME treatment on CsA-induced nitrite and nitrate levels CsA per se caused a significant decrease in urine and tissue nitrite levels while l-arg and molsidomine significantly prevented the decrease in tissue and urine nitrite levels, while pre-treatment with l-NAME prevented this increase in nitric oxide levels (Fig. 1A and B.). 4.4. Effect of l-arginine, molsidomine and l-NAME treatment on CsA-induced lipid peroxidation Chronic CsA treatment caused significant increase in MDA levels as compared to control rats. Concomitant treatment with l-arg as well as with molsidomine significantly prevented the increase in the MDA level, which was reversed by pre-treatment with l-NAME (Fig. 2). 4.5. Effect of l-arginine, molsidomine and l-NAME treatment on CsA-induced alterations in renal antioxidant enzymes Fig. 3 shows the effect of l-arginine, molsidomine and l-NAME pre-treatment on CsA-induced changes in reduced glutathione, SOD and catalase enzyme activities in kidney homogenate. CsA administration caused a significant decrease in reduced GSH, SOD and catalase activities in tissue homogenates. Pre-treatment with l-arg anf molsidomine significantly prevented this fall in renal antioxidant enzyme activities. 4.6. Effect of l-arginine, molsidomine and l-NAME treatment on CsA-induced renal morphological changes The renal morphological changes observed were scored and summarized in Table 3. The light microscopic findings of kidneys of control rats treated with

olive oil for 21 days showed normal glomeruli, afferent arterioles and tubule cells (Fig. 4A). By contrast, the kidneys of rats treated with CsA showed marked histological changes in the cortex and outer medulla. The sections showed severe epical blebbing, hyaline casts and glomerular basement thickening. A marked tubulointerstitial fibrosis of stripped pattern in the cortex (Fig. 4B) and arteriolopathy with hyaline deposition within the tunica media of afferent arteriole and terminal portions of the interlobular arteries and terminal portions of the interlobular arteries were also observed. Co-administration of l-arg and molsidomine showed normal glomeruli, afferent arterioles and tubule cells with mild epical blebbing and hyaline casts (Fig. 4C–E). Cotreatment of these agents with l-NAME prevented this attenuation of renal morphological damage (Fig. 4F–H) (Table 3).

5. Discussion The present investigation revealed that administration of CsA (20 mg/kg, s.c. for 21 days) resulted in an overt nephrotoxicity as evidenced by marked renal dysfunction and significant increase in blood pressure. In addition, the tissue and urine nitric oxide levels were markedly reduced after 21 days alongwith significant depletion of renal oxidative enzymes. These findings were further confirmed by renal morphology. The mechanisms involved in CsA-induced vasoconstriction have not been completely elucidated. Previous studies have shown that administration of the drug stimulates the production of vasoconstrictor factors such as endothelin, thromboxane A2 and angiotensin II (Perico et al., 1986a,b; Kon et al., 1990). However, participation of NO, an important renal vasodilator that maintains the low vascular resistance in kidney, is not well defined. Studies on endothelial cell cultures showed that exposure of cells to CsA results in structural damage (Zoja et al., 1996) and several in vitro studies reported that acetylcholine-induced vasodilation is impaired in vascular beds of CsA-treated animals, suggesting a deficient endothelial NO synthesis (Vaziri et al., 1998; Gossmann et al., 2001), although these findings can also be explained by enhanced generation of free radicals that inactivates NO (Diederich et al., 1994).

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To study whether the CsA-induced changes are related to the hemodynamic effects of this immunosuppressive drug, we evaluated the effect of the concomitant administration of the NO precursor, l-arg and a NO donor, molsidomine. Our results showed that larg as well as molsidomine was able to prevent renal dysfunction as well as changes in tissue and urine nitrite levels, along with the renal oxidative stress induced by CsA. The protection afforded by these agents was blocked by co-treatment with l-NAME, a non-selective NOS inhibitor. Some findings of this study are in concurrence with the previous reports demonstrating that exogenous supplementation of l-arg is effective in reducing renal damage induced by CsA, possibly through the NO pathway (Amore et al., 1995; Yang et al., 1998). In addition, Assis et al. (1997) and De Nicola et al. (1993) showed that oral supplementation of l-arg prevents nephrotoxicity induced by chronic administration of CsA due to formation of more NO, which may enhance vasodilation and consequently reduce the kidney function impairment. Oriji and Keiser (1998) repotted that CsA administration inhibits the endothelial NOS and this inhibition can be overcome by parenteral administration of l-arg. Therefore, the protective effect of l-arg against kidney dysfunction may be related to its reported vasodilatory effect. However, the results of present study revealed that l-arg prevents CsA-induced lipid peroxidation and the significant decrease in renal antioxidative enzymes. Thus it seems that the protective effect induced by l-arg against CsA nephrotoxicity may involve an additional non-homodynamic cytoprotective effect. However, it is difficult to assess, which of these properties is responsible for the protective effect induced by l-arg. In addition, we showed that larginine had a beneficial effect at a much lower dose, as compared to the dose used in previously reported studies. The present investigation also revealed that CsA nephrotoxicity was coupled with significant weight loss, increase in SBP and a marked reduction in tissue and urine nitrite levels and also by the histological studies. In CsA-treated animals, l-arg as well as molsidomine not only prevented renal dysfunction but also abrogated the fall in total tissue and urine NO levels as well as the impairment in the renal oxidative stress. These findings were further confirmed by the histopathological studies. The animals with l-arg and molsidomine treatment showed a significant im-

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provement in renal morphology as compared to CsAtreated animals. The protection afforded by l-arg was attenuated by pre-treatment with l-NAME, indicating that NO is involved in the protection afforded by this agents in cyclosporine nephrotoxicity. Surprisingly, the protective effect observed with molsidomine was also blocked by l-NAME, but this effect may not be through the NOS inhibition. Recently it has been reported that molsidomine also decreases the activity of NOS in vascular wall (Kristek et al., 2003). It is therefore possible that overproduction of NO or excessive levels of NO act as a negative feedback modulator of NOS activity. In the combined l-NAME and molsidomine group, lNAME inhibits NOS, thereby compromising endogenous NO synthesis. Exogenous NO donated by molsidomine may be getting inactivated by CsA-induced O2 •− . This may explain why we have observed no protective effect of molsidomine in the presence of lNAME. In conclusion, the results of the present study indicate that NO plays a pivotal role in CsA nephrotoxicity and administration of molsidomine as well as l-arginine can be useful as a post transplant treatment. Acknowledgements The Senior Research Fellowship of the Council of Scientific and Industrial Research (CSIR), New Delhi, is gratefully acknowledged. The gift sample of cyclosporine by Panacea Biotech Ltd. is gratefully acknowledged. References Amore, A., Gianoglio, B., Ghigo, D., Peruzzi, L., Porcellini, M.G., Bussolino, F., Costamagna, C., Cacace, G., Picciotto, G., Mazzucco, G., 1995. A possible role for nitric oxide in modulating the functional cyclosporine toxicity by arginine. Kidney Int. 47, 1507–1514. Assis, S.M., Monteiro, J.L., Seguro, A.C., 1997. l-Arginine and allopurinol protect against cyclosporine nephrotoxicity. Transplantation 63, 1070–1073. Bohn, H., Schonafinger, K., 1989. Oxygen and oxidation promote the release of nitric oxide from sydnonimines. J. Cardiovasc. Pharmacol. 11, S6–S12. Busauschina, A., Schnuelle, P., vander Woude, F.J., 2004. Cyclosporine nephrotoxicity. Transplant Proc. 36, 229S–233S. Claiborne, A., 1985. In: Greenwald, R.A. (Ed.), CRC Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL, pp. 283–290.

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