Abstract
It was demonstrated that fenofibrate and telmisartan exerted renoprotective effects in ischemia/reperfusion (I/R) injury. Because the combination of fenofibrate and telmisartan synergistically enhanced peroxisome proliferator-activated receptor (PPAR) activation, we hypothesized that the combination of both drugs may exert prolonged beneficial effects in renal I/R injury than fenofibrate alone. Forty-eight male Wistar albino rats were divided into eight groups. Hyperlipidemia was induced by cholesterol feeding for 4 weeks. At the end of the fourth week, renal I/R injury was performed by occlusion of both renal vascular pedicles for 60 minutes, followed by 24 hours of reperfusion. In the treatment group, fenofibrate alone and in combination with telmisartan was administered 2 weeks prior to renal ischemia. At the end of the experiment, blood and kidneys were isolated for biochemical and histological analysis. I/R in hyperlipidemic rat shows significantly increased lipid peroxidation, nitric oxide, and myeloperoxidase activity, and depletion of antioxidant enzyme compared with control rats, and that was significantly restored after fenofibrate and telmisartan treatment. Also, significant increases in serum homocysteine level were detected following I/R. Fenofibrate treatment further elevated homocysteine level, which was reduced by telmisartan in combination with fenofibrate. The most significant histological damage was found in the hyperlipidemic rat subjected to renal I/R, which was reduced significantly with combination therapy. The results of this study concluded that fenofibrate alone and in combination with telmisartan significantly ameliorated renal I/R injury. The additive beneficial effect of telmisartan is predicted to reduce homocysteine-induced oxidative stress through reduced nitric oxide production during I/R.
INTRODUCTION
Hyperlipidemia is one of the major risk factors for cardiovascular disease like atherosclerosis, which may ultimately lead to end-stage renal disease (ESRD).Citation1 Hyperlipidemia increases the production of oxidative stress and inflammatory responses, which may lead to renal cell death.Citation2 With increasing duration and severity of ischemia, however, greater cell damage can develop, with a predisposition to a spectrum of reperfusion-associated pathologies, collectively called reperfusion injury.Citation3 A recent study demonstrated higher incidence of ischemic renal damage in hyperlipidemic rat but the mechanisms are unclear.Citation4 Renal ischemia/reperfusion (I/R) injury initiates a complex series of cellular events that eventually lead to renal cell death. The exact molecular mechanisms underlying renal I/R injury are not fully understood. Several factors contribute to the pathogenesis of I/R injury, including ATP depletion, phospholipase, protease activation, increased endothelin-1 formation, and neutrophil infiltration;Citation5 however, renin–angiotensin system plays an important role in the pathogenesis of renal I/R injury. Angiotensin II (Ang-II), the central product of this system, is involved in oxidative stress and inflammation in renal tissue. Ang-II, by activating the Ang-II type 1 (AT1) receptor, increases generation of reactive oxygen species (ROS) and proinflammatory mediators, particularly tumor necrosis factor-α (TNF-α), which are greatly responsible for renal damage induced by I/R.Citation6,Citation7 Also, I/R impairs renal homocysteine metabolism leading to homocysteine accumulation in the kidney, which contributes to I/R-induced oxidative stress with renal cell death.Citation8
Peroxisome proliferator-activated receptor (PPAR)-α is one of the three subtypes of the nuclear receptor PPAR family, which can be activated by natural ligands, such as polyunsaturated fatty acids, and synthetic ligands. PPARs are implicated in several physiological processes, such as regulation of lipoprotein, lipid metabolism, and glucose homeostasis. In addition to their broad use as lipid-lowering drugs, PPAR-α activation stimulates the expression of antioxidant enzymes, such as superoxide dismutase (SOD) and reduced glutathione (GSH), both of which are involved in renal ischemic injury.Citation9 The selective PPAR-α agonist, fenofibrate, attenuates renal I/R injury,Citation10 but the mechanisms of renoprotective effects have not been investigated.
Telmisartan is a highly selective AT1 receptor antagonist approved for the treatment of hypertension. The renoprotective effect of telmisartan was also demonstrated in spontaneously hypertensive rats,Citation11 partially nephrectomized rats,Citation12 and cyclosporine A-induced nephrotoxicity in pigs.Citation13 In addition, telmisartan possesses antioxidant and anti-inflammatory effects independent of AT1 receptor-blocking activity.Citation14,Citation15 However, telmisartan having PPAR-γ modulating activity produces additional antioxidant and anti-inflammatory effects.Citation14,Citation16
This encouraged us to conduct this study to evaluate the protective effect of fenofibrate alone and in combination with telmisartan against I/R-induced renal injury.
MATERIAL AND METHODS
Animals
Male Wistar albino rats weighting 180–200 g were placed in a temperature (21 ± 2°C) and humidity (60 ± 5%) controlled room in which a 12–12 hours light–dark cycle was maintained. All experiments in this study were performed in accordance with the Committee for Purpose of Control and Safety of Experimental Animals (CPCSEA) guideline and were approved by Institutional Animal Ethical Committee (IAEC).
Drugs
Fenofibrate (Alembic Ltd., Baroda, Gujarat, India) was suspended in 0.5% methyl cellulose solution. Telmisartan (Alembic Ltd.) was prepared in the given 1% aqueous solution of Tween 80. The dose of fenofibrateCitation17 and telmisartanCitation18 used in the work was selected based on the previous study.
Experimental design
Forty-eight rats were randomly divided into eight groups (n = 6): control, hyperlipidemic (HC), renal I/R injury (I/R), HC + I/R, I/R + fenofibrate (I/R + FF), I/R + fenofibrate + telmisartan (I/R + FF + TS), HC + I/R + FF, and HC + I/R + FF + TS groups. Cholesterol (Sigma Chemical Co., St. Louis, MO, USA), in hydrogenated groundnut oil (as a vehicle), was administered orally at a dose of 500 mg/kg/day.Citation19 Control animals were administered with the vehicle alone. Development of hyperlipidemia was confirmed by measuring serum total cholesterol level 2 weeks after starting of cholesterol administration. In HC groups (24 rats), rats with serum total cholesterol levels of 200 mg/dL or above were considered to be hyperlipidemic. Starting on the same day of the diagnosis of hyperlipidemia, fenofibrate (100 mg/kg/day, p.o.) alone and in combination with telmisartan (0.3 mg/kg/day, i.p.) was administered for consecutive 2 weeks. The rats were anesthetized with ketamine hydrochloride (60 mg/kg, i.p.) and diazepam (5 mg/kg, i.p.), and surgical procedure was performed as previously described.Citation20 Briefly, a midline incision was made and both renal pedicles were exposed and clamped using fine vascular clips. The rats were kept on a heat pad and the temperature was maintained at 37°C throughout the experimental procedure. The renal vascular pedicles were occluded for 60 minutes (ischemic phase), after which the clips were removed to start the reperfusion phase for 24 hours. After the surgical procedures, the midline incision was sutured back with the local applications of povidone and Neosporin. The animals were allowed to recover from anesthesia. The rats were killed after completion of I/R and kidneys were quickly removed and placed into liquid nitrogen and then stored at –70°C until assayed for biochemical parameters. Blood samples were collected before the animals were killed and centrifuged for 10 minutes at 2800 g. The obtained clear sera were stored at –20°C for subsequent measurement of lipid profile, serum creatinine, and urea levels using semiautomated biochemistry analyzer-photometer 5010 (Piramal Healthcare, Mumbai, India) using a commercially diagnostic kit purchased from Piramal Healthcare.
Tissue preparations for analysis
After weighing kidney tissue, it was homogenized in ice cold 150 mM KCl solution using homogenizer. The renal homogenates were centrifuged at 2800 g for 10 minutes at 4°C. The resulting supernatant was used for determination of malondialdehyde (MDA), GSH, metabolic end products of nitric oxide (NO) (nitrate and nitrites) levels, and myeloperoxidase (MPO) activity.
Determination of MDA level
The tissue MDA level was determined by a previously described method based on the reaction with thiobarbituric acid (TBA) at 90–100°C. In the TBA test reaction, MDA or MDA-like substances and TBA react with the production of a pink pigment having an absorption maximum at 532 nm. The reaction was performed at pH 2–3 at 90°C for 15 minutes. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid to precipitate protein. The pellet was precipitated by centrifugation, and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in boiling water bath for 10 minutes. After cooling, the absorbance was read at 532 nm.Citation21
Determination of GSH level
The tissue GSH was determined by the spectrophotometric method with Ellman's procedure.Citation22 Briefly, after centrifugation at 2000 × g for 10 minutes, 0.5 mL of supernatant was added to 2 mL of 0.3 mol/L Na2HPO4·2H2O solution. A 0.2-mL solution of dithiobisnitrobenzoate (0.4 mg/mL 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. GSH levels were calculated using an extinction coefficient of 1.36 × 105 M−1 cm−1.
Determination of metabolic end products of nitric oxide (nitrate and nitrites) level
Renal NO level was estimated by the method described by Guevara et al.Citation23 Samples were initially deproteinized with Somogyi reagent. Total nitrite (nitrite + nitrate) was measured by spectrophotometry at 545 nm after conversion of nitrate to nitrite by copperized cadmium granules. A standard curve was established with a set of serial dilutions of sodium nitrite. The resulting equation was used to calculate the unknown sample concentrations.
Determination of MPO activity
Renal MPO activity was measured in renal tissues as described by Wei et al.Citation24 It was determined using a 4-aminoantipyrine/phenol solution as the substrate for MPO-mediated oxidation by H2O2 and changes in absorbance at 510 nm were recorded. One unit of MPO activity is defined as that which degrades 1 μmol H2O2/min at 25°C.
Determination of homocysteine level
Homocysteine levels were then measured in the serum samples according to the instructions of the manufacturer of the homocysteine reagent kit (Chromsystems, Munchen, Germany).
Histopathological evaluation
The kidneys fixed in 10% neutral-buffered formalin solution were embedded in paraffin and were used for histopathological examination. Sections of 5 μm thickness were cut in a microtome and taken on glass slides coated with albumin. The hematoxylin-stained sections were stained with eosin for 2 minutes and were then quickly passed through ascending grades of alcohol, cleaned in xylene, and mounted on Canada Balsam. The stained sections were examined under Olympus BX40 photomicroscope and photographed. Either samples were coded to perform blind study or expert guidance from the veteran pathologist was sought to determine histopathological changes. 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.
Statistical analysis
All the values are expressed as mean ± SD. Statistical significance between more than two groups were tested using one-way analysis of variance (ANOVA) followed by the Bonferroni posttest. Differences were considered to be statistically significant when p < 0.05.
RESULTS
Kidney function
HC as well as non-HC rats subjected to renal I/R resulted in significant rises in serum creatinine and urea levels as compared to the control and HC group. Fenofibrate at the dose of 100 mg/kg/day for pre-ischemic treatment for two consecutive weeks significantly reduced serum creatinine and urea level as compared with I/R and HC + I/R group. Telmisartan (0.3 mg/kg/day) along with fenofibrate (100 mg/kg/day) treatment for two consecutive weeks significantly restored serum creatinine and urea at normal level ().
Table 1. Serum creatinine, urea, and homocysteine levels of all experimental groups
Renal oxidative stress markers
HC as well as non-HC rats subjected to I/R injury produced significant increase in renal MDA content, but decrease in renal GSH activity, when compared to control rats. Fenofibrate treatment significantly prevented the depletion of the antioxidant defense mechanism in kidney tissue resulting from I/R in HC as well as non-HC rats. Also, the increased renal MDA level induced by I/R was significantly suppressed by fenofibrate treatment. Combination of fenofibrate and telmisartan significantly increase the GSH level and restored MDA at normal level ().
Table 2. Renal MDA, GSH, NO, and MPO levels of all experimental groups
Renal inflammatory markers
The levels of the inflammatory markers, renal NO, and MPO in renal homogenates were significantly elevated in HC as well as in non-HC rats exposed to I/R as compared to respective control groups. Pretreatment with fenofibrate significantly reduced I/R-induced elevations in renal NO and MPO levels. Telmisartan in combination with fenofibrate restored the normal level of renal NO and MPO level when compared to control rats ().
Serum homocysteine levels
Renal I/R injury in HC as well as in non-HC rats resulted in significant increases in serum levels of homocysteine as compared to the corresponding values in the control groups. Two consecutive weeks of fenofibrate treatment further elevated homocysteine level in both I/R and HC + I/R groups. Telmisartan in combination with fenofibrate restored the normal level of serum homocysteine when compared to control groups ().
Histological examination
Histological examination of the kidneys showed that HC as well as non-HC rats subjected to I/R caused severe and widespread necrosis with dilatation, vacuolar degeneration, epithelial desquamation, and intra-luminal cast formation mainly in the proximal convoluted tubules, whereas the normal structure of the glomeruli was preserved. However, telmisartan along with fenofibrate treatment resulted in marked attenuation of tubular necrosis and dilatation, with absence of vacuolization and cast formation induced by I/R in HC as well as in non-HC rats ().
Table 3. Morphological changes of kidney tissue in all experimental groups
DISCUSSION
This work, in agreement with previous studies, clearly demonstrated that increased MDA level, depletion of antioxidant defense mechanism, and the release of proinflammatory mediators are greatly responsible for renal injury mediated by I/R in normal as well as in HC rats.Citation2,Citation25–27 In this study, pretreatment with fenofibrate alone and in combination with telmisartan for two consecutive weeks effectively protected against acute I/R-induced renal injury in HC as well as in non-HC rats, as indicated by the significant improvement in the disturbed biochemical parameters and marked amelioration of renal tissue damage observed by histological examinations.
Activation of the renin–angiotensin pathway with increased production of Ang-II plays an important role in the pathogenesis of renal I/R injury.Citation6,Citation7 Ang-II is a well-known oxidative stress inducer, and ROS are involved in many of the Ang-II signaling pathways. Ang-II, through the AT1 receptor, stimulates the NADPH oxidase enzyme with increased generation of superoxide anion, hydrogen peroxide, and hydroxyl radicals.Citation28 Generation of ROS during I/R activates inflammatory cascades with increased production of proinflammatory cytokines leads to neutrophil infiltration into inflammatory organs is responsible for renal tissue injury.Citation25,Citation29,Citation30 In accordance with these results, previous studies demonstrated that acute renal I/R caused intense inflammatory reaction with overexpression of TNF-α and NO in the HC kidney tissue. This can be explained by rapid upregulation of mRNA for TNF-α and inducible nitric oxide synthase observed during I/R.Citation2,Citation27,Citation29 Excess NO exerts toxicological effects such as vasodilation, edema, cytotoxicity, and mediation of cytokine-dependent processes. It reacts with superoxide anion to generate the peroxynitrite radical that causes further cell damage. Also, excess NO depletes intracellular GSH increasing the susceptibility to oxidative stress.Citation31,Citation32
Fenofibrate, PPAR-α agonist, was proved effective in reducing the generation of ROS, overexpression of TNF-α, and excess NO production observed with oxidative and inflammatory tissue injuries.Citation10,Citation17,Citation33 The antioxidant and anti-inflammatory effects of fenofibrate are related to its ability to suppress the protein expression for NADPH oxidase, TNF-α, and inducible nitric oxide synthase by inhibiting the phosphorylation of p38 MAPK (mitogen-activated protein kinase). Fenofibrate, therefore, prevents the activation of the nuclear factor-κB signaling pathway, which promotes the transcription of NADPH oxidase, TNF-α, and inducible nitric oxide synthase genes.Citation10,Citation33,Citation34 In addition, telmisartan along with fenofibrate protects renal cell by suppressing oxidative stress and proinflammatory mediators. Independent of AT1 receptor-blocking activity, telmisartan is responsible for the additional antioxidant and anti-inflammatory activities. The drug acts as a partial agonist of PPAR-γ.Citation14,Citation15,Citation35,Citation36 Activation of PPAR-γ induces catalase gene expression and inhibits nuclear factor-κB, thus, combating oxidative stress and downregulating most of the proinflammatory responses.Citation37,Citation38 Another possible mechanism is that telmisartan, by blocking AT1 receptor, allows Ang-II to interact more with the Ang-II type-2 receptor (AT2) which, on the contrary, provides cellular protective action against oxidative stress and excitotoxic damage.Citation39,Citation40
Also, recent studies demonstrated that elevated homocysteine level during I/R is attributed to reduced activity of cystathionine-β-synthase (CBS) that catalyzes the rate-limiting step in renal homocysteine metabolism.Citation8 Homocysteine, at elevated levels, is associated with oxidative stress and lipid peroxidation leading to renal cell death.Citation8 It was found that decreasing the NO level prevented the inhibition of CBS enzyme in the kidneys subjected to I/R. This led to the conclusion that excess nitric oxide production in the reperfusion phase is the factor most probably responsible for the reduced CBS activity.Citation41 This study showed that fenofibrate treatment further elevated I/R-induced homocysteine levels. The underlying mechanism for the increase of homocysteine after fenofibrate therapy has not yet been revealed. Dierkes et al. showed that there was no change in folate and cobalamin status. Furthermore, serum cystatin C, an indirect marker of renal function, increased significantly after fenofibrate therapy, which suggests that impaired renal function caused the increase in total serum homocysteine.Citation42 However, in addition to the confirmed increase in serum creatinine after fenofibrate, we saw decreased serum creatinine concentration, indicating no change in renal function. Another, unstudied, explanation for the increase in serum total homocysteine could be the involvement of PPAR-α. Fenofibrate exerts its effect through the activation of PPAR-α.Citation43,Citation44 Because activation of PPAR-α results in altered genetic regulation of various metabolic pathways, it may also lead to altered regulation of homocysteine metabolism, resulting in increased serum total homocysteine. Telmisartan in combination with fenofibrate significantly reduced homocysteine up to the normal level. This can be considered as another possible mechanism for the additive beneficial effect of telmisartan in combination with fenofibrate against the oxidative stress and renal cell death that occur during I/R. It can be speculated that telmisartan prevented homocysteine elevation by restoring the activity of CBS as a result of reduced NO production during I/R. However, this needs to be clarified by further investigation.
The duration of the ischemic phase (60 minutes) and reperfusion phase (24 hours) applied in this work was selected based on a previous study, which demonstrated that the peak elevation of intrarenal Ang-II level leads to the release of proinflammatory cytokines (TNF-α and interleukin-6), and inducible nitric oxide synthase in renal tissue occurred at this time point and decreased gradually over a 120-hour period of reperfusion.Citation20
CONCLUSION
The results of this study concluded that fenofibrate significantly ameliorated renal I/R injury. Suppressed MDA content, MPO activity, improved antioxidant enzyme, and reduced generation of proinflammatory mediators are the main factors contributing to the renoprotective effect of fenofibrate. However, additive beneficial effect of telmisartan is predicted to reduce homocysteine-induced oxidative stress through reduced nitric oxide production during I/R.
Acknowledgment
The authors thank Alembic Ltd., Baroda, Gujarat, India, for supplying fenofibrate and telmisartan as a gift sample.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
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