Abstract
Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The causes for the nephrotoxicity of CsA have not been fully elucidated. Intrarenal vasoconstriction induced by several different mediators, both in humans and experimental animals, have been proposed. To determine if the renal alterations are mediated directly by cyclosporine or by secondary hemodynamic alterations induced by cyclosporine, we evaluated if nifedipine prevents 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/mL, s.c. for 21 days), groups 3, 4, and 5 received CsA along with various doses of nifedipine (5, 10, and 20 mg/kg, p.o.) 24 h before and 21 days concurrently, groups 6, 7, and 8 received L-NAME (10 mg/kg i.p.), propranolol (10 mg/kg i.p.), and aminoguanidine (100 mg/kg p.o.), respectively, along with CsA. 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 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, and significantly deranged the renal functions as well as renal morphology. Treatment with nifedipine (10, 20 mg/kg) significantly improved the renal dysfunction, tissue and urine total nitric oxide levels, and renal oxidative stress and prevented the alterations in renal morphology. These results clearly demonstrate that nifedipine is beneficial as a protective agent against nephrotoxicity induced by CsA, and the protection afforded by nifedipine appears to be mediated by an increase in endothelial nitric oxide release.
Introduction
Cyclosporine (CsA) is an important immunosuppressive agent widely used to prevent the rejection of transplanted organs and to treat autoimmune diseases. However, the use of this drug is complicated by diverse side effects like hypertension and nephrotoxicity, which is most common and grave.Citation[1&2] CsA nephrotoxicity is characterized by intense renal vasoconstriction that induces a fall in renal blood flow and in the glomerular filtration rate. This side effect is usually reversible when the CsA dose is reduced. However, even with normal blood levels or during long-term dosage, CsA can produce chronic hypertension and can be the cause of irreversible impairment of renal function.Citation[3] Renal vasoconstriction is attributed to an imbalance in the release of vasoactive substances: on one hand, increased release of vasoconstrictive factors such as thromboxane,Citation[4] endothelin,Citation[5] and angiotensin II;Citation[6] and, on the other, a decrease in vasodilating factors such as prostacyclinCitation[6] and nitric oxide (NO).Citation[7&8]
Renal dysfunction can occur at any time and ranges from an early reversible damage to a late progression to irreversible chronic renal failure. Acute nephrotoxicity may appear soon after transplantation or after weeks or months, with oliguria, acute decrement of glomerular filtration rate and renal plasma flow. Morphological changes to the kidney, consisting of tubular diffusion vacuolization, single-cell necrosisCitation[9] and microcalcification are fully reversible.Citation[10] Experimental studies in spontaneously hypertensive rats revealed a nephrotoxicity similar to that observed in man.Citation[11] Furthermore, hemolytic uremic syndrome and irreversible functional and morphological changes have been induced by this agent when it was administered for long periods of time.Citation[10]
In the kidney, NO is a vasoactive factor that plays a key role in maintaining vascular tone. NO is produced from l-arginine by the action of nitric oxide synthase (NOS) isoforms, of which at least three molecular-level isoforms have been identified. All three NOS isoforms are present in the kidney. In renal cortex, nNOS exhibits a macula densa cell-specific expression, and iNOS has been observed in mesangial and proximal tubule cells of the afferent and efferent arterioles and glomerular capillaries. Under pathological conditions, iNOS catalyses an adequate quantity of inducible NO.Citation[12&13] It has been shown that the intense vasoconstriction induced by CsA administration is associated with increased eNOS mRNA in renal cortex and decreased nNOS and iNOS mRNA in renal medulla,Citation[14&15] suggesting that the effect of CsA on NOS expression could be tissue specific. In addition, it is well documented that eNOS expression can be activated by shear stress,Citation[16&17] which may result from vasoconstriction. Thus, hemodynamic changes induced by CsA could be the major mechanism by which this immunosuppressive drug alters NO levels.
With this background, the present study was designed to investigate the possible protective effect of a vasodilator agent nifedipine on changes in nitric oxide levels, renal oxidative stress, and nephrotoxicity induced by CsA. Furthermore, because NO is rapidly deactivated by reactive oxygen species (ROS),Citation[18] and dihydropyridine (DHPs) may act as scavengers as known from different in vitro models,Citation[19-21] we also determined the antioxidative potency of nifedipine by measuring the levels of different antioxidative enzymes to reveal a potential NO-protection effect as an underlying mechanism of the increased NO bioavailability.
Materials and Methods
Chemicals
Cyclosporine was used in powder form (gift from Panacea Biotech Ltd., Lalru, India), nifedipine, (Unique Pharmaceuticals, Mumbai, India). All the remaining chemicals were of highest commercially available grade.
Animals
Male Wistar rats (150 g–200 g), bred in the central animal house of Punjab 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 Punjab University, Chandigarh.
Experimental Groups
Animals were distributed into eight groups, each comprised of six to eight animals. The doses of nifedipine, CsA, L-NAME, propranolol, and aminoguanidine were selected on the basis of extensive literature survey and from the preliminary studies performed in our laboratory.
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. The second group received CsA (20 mg/mL, s.c.) dissolved in olive oil for 21 days. The rats in the third group received nifedipine (5 mg/kg, p.o.) 24 h before administering CsA, and continued with CsA for 21 days. The rats in the fourth group received a dose of nifedipine (10 mg/kg, p.o.) 24 h before administering CsA, and continued with CsA for 21 days. The rats in the fifth group received a dose of nifedipine (20 mg/kg, p.o.) 24 h. before administering CsA, and continued with CsA for 21 days. The rats in the sixth group received a dose of L-NAME, a nonselective NOS inhibitor (10 mg/kg, i.p.) along with CsA for 21 days. The rats in the seventh group received a dose of propranolol (10 mg/kg, i.p.) along with CsA for 21 days. The rats in the eighth group received a dose of aminoguanidine, a specific iNOS inhibitor (100 mg/kg, p.o.), along with CsA for 21 days. 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 h after the last dose for urine collection. On day 22, animals were anesthetized with thiopentone sodium (60 mg/kg, i.p.), and blood was collected through the 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 until further enzymatic analysis, whereas, the right kidney was stored in 10% formalin for the histological studies.
Assessment of Renal Function
Plasma samples were assayed for blood urea nitrogen (BUN), urea clearance, serum creatinine, and creatinine clearance by using standard diagnostic kits (Span Diagnostics, Gujarat, India).
Estimation of Tissue and Urine Nitrite and Nitrate Levels
Nitrite and nitrate are the primary oxidation products of NO subsequent to reaction with oxygen; 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 naphthyl ethylenediamine and sulfanilamide. 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 µmol/L.
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).Citation[22] In brief, the reaction mixture consisted of 0.2 ml of 8.1% sodium lauryl sulfate, 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 postmitochondrial supernatant (PMS). The mixture 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 milligram protein. Tissue protein was estimated using Biuret methodCitation[23] of protein assay, and the renal MDA content was expressed as nanomoles of malondialdehyde per milligram of protein.
Assessment of Antioxidant Enzymes (AOE)
The kidney homogenate was used to assay reduced glutathione (GSH), catalase, and superoxide dismutase (SOD) activity.
The AOE were estimated by the well-established procedures already published elsewhere.Citation[24] The nonprotein sulfhydryl (NPSH) as a marker for reduced glutathione (GSH) was measured by the method of Jollow et al.,Citation[25] and the yellow color developed by the reduction of Ellman's reagent by –SH groups of NPSH was read at 412 nm. The catalase (CAT) activity was assayed by the method of Claiborne,Citation[26] and the rate of decomposition of H2O2 was followed at 240 nm. The superoxide dismutase (SOD) activity was assessed by the method of Kono.Citation[27] The nitro blue tetrazolium (NBT) reduction by superoxide anion to blue formazan was followed at 560 nm.
Renal Histology
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 solution, embedded in paraffin, and used for histopathological examination. Sections that were 5 µm thick 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 of 10 fields for each kidney slide was examined and assigned for severity of changes using scores on a scale of none (−), mild (+), moderate (++), and severe (+++) damage.
Statistical Analysis
The data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test for comparing means from different treatment groups. The data were expressed as mean ± SEM, and a value of p < 0.05 was considered statistically significant.
Results
Effect of Nifedipine 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 nifedipine (10 and 20 mg/kg). CsA administration per se caused a significant rise in SBP. The animals receiving nifedipine (10 and 20 mg/kg, p.o.) and propranolol (10 mg/kg, i.p.) along with CsA reduced this rise in SBP; however, animals receiving L-NAME along with CsA further showed an increase in SBP and weight loss ().
Table 1. Effect of nifedipine (5, 10, and 20 mg/kg) treatment on body weight and SBP in chronic CsA (20 mg/kg) treated rats.
Effect of Nifedipine Treatment on CsA-Induced Renal Dysfunction
demonstrates the effect of nifedipine on renal dysfunction induced by CSA (20 mg/ml s.c. for 21 days). CsA administration caused abnormal renal function in all rats. Serum creatinine and BUN were significantly increased in comparison to control rats. Pretreatment of animals with nifedipine (10 and 20 mg/kg, p.o.) reduced the rise in the levels of serum creatinine and BUN. There was a significant decrease in creatinine and urea clearance in CsA-treated rats, which was markedly improved by concomitant treatment with nifedipine. However, L-NAME, AMG, and propranolol pretreatment did not attenuate renal dysfunction induced by CsA (, , , ).
Figure 1 Effect of nifedipine (5, 10, and 20 mg/kg) on serum creatinine (A), blood urea nitrogen (BUN) (B), creatinine clearance (C), and urea clearance (D) in CsA treated rats. 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 < 0.01 as compared to NFI(10) + CsA group (one-way ANOVA followed by Dunnett's test).
Effect of Nifedipine Treatment on CsA-Induced Nitrite and Nitrate Levels
CsA caused a significant decrease in urine and tissue nitrite levels, while nifedipine administration (10 and 20 mg/kg) significantly prevented the decrease in tissue and urine nitrite levels; however, L-NAME treatment further decreased the urine and tissue total nitric oxide levels. Treatment with AMG and propranolol did not improve this decrease in tissue and urine nitric oxide levels (, ).
Figure 2 Effect of nifedipine (5, 10, and 20 mg/kg) on CsA-induced urine and tissue nitrite levels (A, 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 < 0.01 as compared to NFI(10) + CsA group (one-way ANOVA followed by Dunnett's test).
Effect of Nifedipine Treatment on CsA-Induced Lipid Peroxidation
Chronic CsA treatment caused a significant increase in MDA levels as compared to control rats. Concomitant treatment with nifedipine (10 and 20 mg/kg) significantly prevented the increase in the MDA level, which was not the case in L-NAME, AMG, and propranolol treated animals ().
Figure 3 Effect of nifedipine (5, 10, and 20 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 < 0.01 as compared to NFI(10) + CsA group (one-way ANOVA followed by Dunnett's test).
Effect of Nifedipine Treatment on CsA-Induced Alterations in Renal Antioxidant Enzymes
shows the effect of nifedipine pretreatment 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. Pretreatment with nifedipine (10 and 20 mg/kg, p.o.) significantly prevented this decrease in renal antioxidant enzyme activities; however animals treated with L-NAME, AMG, and propranolol did not improve this decrease in antioxidant enzyme levels.
Table 2. Effect of nifedipine (5, 10, and 20 mg/kg) treatment on renal antioxidant enzymes in chronic CsA (20 mg/kg) treated rats.
The Effect of Nifedipine Treatment on CsA-Induced Renal Morphological Changes
The renal morphological changes observed were scored and summarized in . The light microscopic findings of kidneys of control rats treated with olive oil for 21 days showed normal glomeruli, afferent arterioles, and tubule cells (A). 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 moderate interstitial vacuolization of the distal tubules and glomerular basement thickening. A marked tubulointerstitial fibrosis of stripped pattern in the cortex (B) 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 (C). Coadministration of nifedipine (10 and 20 mg/kg) showed normal glomeruli, afferent arterioles, and tubule cells with mild epical blebbing and hyaline casts (E, F). Treatment of animals with L-NAME further worsened the histological injury observed with CsA treatment; however, AMG and propranolol treated animals revealed similar degrees of damage as that of CsA-treated animals ().
Table 3. Effect of nifedipine (5, 10, and 20 mg/kg) treatment on morphological changes as assessed by histopathological examination of kidney in CsA treated rats.
Figure 4 Hematoxylin and eosin stained sections of rat kidneys: A) renal cortex of rat treated with vehicle (olive oil); B) interstitial fibrosis of stripped pattern and tubular atrophy in cortex of CsA-treated rats; C) arteriole with marked luminal narrowing and pronounced intimal thickening in CsA rats; D) renal cortex of rats treated with nifedipine (5 mg/kg) showing interstitial fibrosis as well as arteriolopathy similar to CsA; E) renal cortex of rats treated with nifedipine (10 mg/kg) showing some arteriolopathy, interstitial fibrosis; and F) renal cortex of rats treated with nifedipine (20 mg/kg) that prevented the development of CsA-induced alterations.
Discussion
CsA administration for 21 days resulted in a significant increase in blood pressure, derangement of renal function, renal oxidative stress, as well as a marked decrease in tissue and urine nitric oxide levels. 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 vasoconstrictive factors such as endothelin, thromboxane A2, and angiotensin II.Citation[4-6] 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,Citation[28] 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,Citation[7&8] although these findings can also be explained by enhanced generation of free radicals that inactivates NO.Citation[29] Recent studies demonstrated that exogenous supplementation of l-arginine is effective in reducing renal damage induced by CsA, possibly through the NO pathway, which may enhance vasodilation and, consequently, reduce the renal function impairment.Citation[30-32] Oriji and KeiserCitation[33] reported that CsA administration inhibits the endothelial NOS enzyme, and this inhibition can be overcome by parenteral administration of l-arg.
Dihydropyridine (DHP)-type calcium antagonists are important drugs in the treatment of hypertension and coronary heart disease. They induce their specific pharmacological effects by binding to l-type calcium channels,Citation[34&35] which results in a reduced calcium influx with impaired electrochemical coupling both in vascular smooth muscle cells and in the heart.Citation[36] A few years ago, however, it was observed that removal of the endothelium or blockade of the guanylate cyclase of the vessel wall reduced the efficacy of the DHP-induced vasorelaxation,Citation[37] which indicated an endothelium-responsive cGMP mediated process as part of the DHP action. Because macrovascular endothelial cells voltage-operated l-type calcium channels,Citation[38&39] the DHPs must exert these effects via other mechanisms. With this background, it is tempting to speculate that NO, one of the most prominent endothelium-derived factors,Citation[40&41] which relaxes smooth-muscle cells via the cGMP signal cascade, might be involved in these DHP actions. In fact, in various models, evidence has accumulated that DHPs stimulate endothelial NO release,Citation[42-48] which may mediate or at least contribute to the-above mentioned calcium channel-independent effects. Berkels et al.Citation[48-50] demonstrated that acute exposure to nifedipine in the micromolar and submicromolar concentration ranges stimulates the NO release from the endothelium.
In the present study, we chose to investigate nifedipine because it was previously shown by Naruse et al.Citation[51] that this calcium channel blocker has no effect on the eNOS gene expression in the kidney, and it was shown to produce renal and hepatic vasodilation.Citation[52-55] In CsA-treated animals, nifedipine (10 and 20 mg/kg, p.o.) not only prevented the decrease in renal function but also abrogated the fall in nitrite levels in the tissue and urine as well as the impairment in renal oxidative stress. These findings were further confirmed by the histopathological studies. The animals treated with nifedipine showed a significant improvement in renal morphology as compared to CsA-treated animals. However, the animals treated with L-NAME and propranolol did not attenuate the nephrotoxicity, fall in nitric oxide levels, and oxidative stress induced by CsA. The results of the present study are in harmony with the results of Ferguson et al.Citation[56] whereby nifedipine was shown to abolish the renal arteriolar vasospasm produced by cyclosporine. However, the results of the present study revealed that nifedipine prevents the rise in lipid peroxides, significantly improves renal antioxidative enzyme levels, and leads to an increase in endothelial nitric oxide levels. Treatment of rats with AMG (100 mg/kg p.o.) did not result in any protection against kidney damage, decrease in nitric oxide levels, and oxidative stress induced by CsA. This finding supports the previous report of Wu et al.,Citation[57] who studied the effect of AMG and CsA on iNOS of mouse-cultured mTAL cells and demonstrated that AMG inhibits iNOS in a similar way to inhibition of iNOS by CsA.
In our study, nifedipine markedly reduced oxygen free radical (OFR) production in the CsA-treated rats. The reduction in OFR might have led to reduced inactivation of NO, resulting in elevated levels of NO. In conclusion, the results of the present study may indicate that nifedipine is beneficial as a protective agent against nephrotoxicity induced by CsA, and the protection afforded by nifedipine appears to be mediated by the increase in nitric oxide release or availability due to inactivation by oxygen-free radicals.
Acknowledgments
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.
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