Effect of naringin on hemodynamic changes and left ventricular function in renal artery occluded renovascular hypertension in rats.

BACKGROUND: Renal artery occlusion (RAO) induced hypertension is a major health problem associated with structural and functional variations of the renal and cardiac vasculature. Naringin a flavanone glycoside derived possesses metal-chelating, antioxidant and free radical scavenging properties.
OBJECTIVE: The objective of this study was to investigate the antihypertensive activity of naringin in RAO induced hypertension in rats.
MATERIAL AND METHODS: Male Wistar rats (180-200 g) were divided into five groups Sham, RAO, naringin (20, 40 and 80 mg/kg). Animals were pretreated with naringin (20, 40 and 80 mg/kg p.o) for 4 weeks. On the last day of the experiment, left renal artery was occluded with renal bulldog clamp for 4 h. After assessment of hemodynamic and left ventricular function various biochemical (superoxide dismutase [SOD], glutathione [GSH] and malondialdehyde [MDA]) and histological parameters were determined in the kidney.
RESULTS: RAO group significantly (P < 0.001) increased hemodynamic parameters at 15, 30 and 45 min of clamp removal. Naringin (40 and 80 mg/kg) treated groups showed a significant decrease in hemodynamic parameters at 15 min. after clamp removal that remained sustained for 60 min. Naringin (40 and 80 mg/kg) treated groups showed significant improvement in left ventricular function at 15, 30 and 45 min after clamp removal. Alteration in level of SOD, GSH and MDA was significantly restored by naringin (40 and 80 mg/kg) treatment. It also reduced histological aberration induced in kidney by RAO.
CONCLUSION: It is concluded that the antihypertensive activity of naringin may result through inhibition of oxidative stress.

Cardiovascular diseases (CVD) contemplate as the principal cause of bereavement in the world. Hypertension or simply high blood pressure is the one of the most imperative risk factor accountable for atherosclerosis, coronary artery disease, myocardial infarct, heart failure, stroke and renal insufficiency.[12] In the field of nephrology, renal hypertension is postulated as an instigation of acute renal failure. Kidney plays a fundamental role in the continuance of peripheral vascular resistance through the action of angiotensin-II a final product of renin-angiotensin system (RAS) and entail in the volume control of cardiac output by regulating urinary salt and water excretion. Consequently, RAS plays a fundamental role in the progress of renovascular hypertension (RVHT). Renal hypertension is commenced by physiological variations in the kidney through a weird stipulation. Renal ischemia-reperfusion (I/R) injury is a major burden on both surgeons as well as on patient due to frequent cause of acute renal failure which occur during renal artery surgery, renal transplantation as well as in cardiovascular surgery.[345]

Angiotensin spurs intracellular formation of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide, which eventually mayhem the physiology of the kidney.[6] During renal ischemia-reperfusion (RIR) injury reactive oxygen and nitrogen species played a promising role in mediating cell damage.[78] Furthermore, RIR is a complex pathophysiological process, which is caused by various confrontations like oxygen free radical formation, mitochondrial dysfunction, tubular cell apoptosis and inflammatory cytokines generation.[91011] It has been distinguished that hypertension is the brisk progress of necrosis caused by critical imbalance between the oxygen supply and the demand of the cells. Oxidative stress resulting from augmented production of free radicals allied with reduced levels of antioxidants in the myocardium plays a major role in cardiovascular complications such as ischemic heart disease, atherosclerosis, congestive heart failure, cardiac hypertrophy, and arrhythmias.[12] In addition, generation of toxic ROS such as superoxide radical, hydrogen peroxide, and hydroxyl radical may regard as the principal conjecture for cardiovascular dysfunction.[13] For upholding of customary physiological function free radicals are elementary, however, overabundance or an unconstrained quantity of free radicals instigate as well as promulgate appallingly fatal to the cells.[1415]

In recent times copious of studies substantiate that deployment of diets rich in plant provisions are allied with a truncated risk of cardiovascular compactions.[16] For the treatment of CVD researchers have illustrated rehabilitated curiosity in medicinal plants and food products for their certain preventive measures. Flavonoids are extensively disseminated in vegetables fruits tea and wines which are renowned as naturally demonstrating antioxidants that hamper lipid peroxidation in biological membranes.[17181920] In recent year, flavonoids catch eye of researchers due to strong antioxidant ability of defending human body against oxidative stress and free radical.[2122] Moreover, bioflavonoid played decisive biological role, which include function of scavenging ROS.[2324] It has been acknowledged that diseases associated to oxidative stress can be treated by consumption of dietary antioxidants.[2526272829] It is well-demonstrated that antioxidant defense system is modulated by scavenging free radicals that are known to exert by isolated bioactive moieties.

Currently, there is much interest in the usefulness of citrus fruits because of their intake emerges to be linked with reduced risk of certain chronic diseases and increased endurance.[30] In addition, flavonoids have antioxidant capacities that are much stronger than those of Vitamins C and E.[31] Naringin (4’,5,7-trihydroxyflavanone 7-rhamnoglucoside) is a major and active bioflavonoid occur in grape and citrus fruits with the ability of compelling antioxidative action.[3233] Naringin has various potential pharmacological and therapeutic characteristics such as antiinflammatory, antimutagenic, cholesterol lowering, antiulcer, anticancer, free radical scavenging, neuroprotective, colono-protective and antioxidant action.[1720343536] Naringin exhibits a potential cardio-protective effect through modulation of oxidative stress and inflammatory markers in doxorubicin as well as isoproterenol induced cardiotoxicity.[3738] It has been reported that naringin revealed protective effect on glycerol-induced acute renal failure in rat kidney.[39] It has been supported that naringin exerts its renoprotective effect in ischemia reperfusion injury in rats conceivably due to overwhelming antioxidant proficiency and free radical scavenging activity.[40] Hence, the present investigation is anticipated to appraise antihypertensive potential of naringin in renal artery occluded (RAO) hypertensive rats through assessment of antioxidant motion on assorted enzymes.

Materials and Methods

Experimental animals and research protocol approval

Adult male Sprague-Dawley rats (180-200 g) were purchased from the National Institute of Biosciences, Pune (India). They were maintained at 24°C ± 1°C, with relative humidity of 45-55% and 12:12 h dark/light cycle. The animals had free access to standard pellet chow (Pranav Agro Industries Ltd., Sangli, India) and water throughout the experimental protocol. All experiments were carried out between 09:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee of Poona College of Pharmacy, Pune and performed in accordance with the guidelines of Committee for Control and Supervision of Experimentation on Animals.

Drugs and chemicals

Naringin was purchased from Sigma Chemical Co., (St. Louis, MO, USA). 1,1’,3,3’-Tetraethoxypropane, crystalline beef liver catalase, reduced glutathione (GSH), 5,5’-dithiobis (2-nitrobenzoic acid) were purchased from S. D. Fine Chemicals, Mumbai, India.

Experimental design

Animals were divided randomly into five groups 12 animals each as follows.

Group I: Sham: Rats were exposed to left renal artery but did not receive any occlusion. They were treated with distilled water (10 mg/kg)

Group II: RAO control: Rats were exposed to left renal artery undergoes RAO. They were treated with distilled water (10 mg/kg)

Group III: RAO + N (20): Rats were exposed to left renal artery undergoes RAO. They were received naringin 20 mg/kg, p.o

Group IV: RAO + N (40): Rats were exposed to left renal artery undergoes RAO. They were received naringin 40 mg/kg, p.o

Group V: RAO + N (80): Rats were exposed to left renal artery undergoes RAO. They were received naringin 80 mg/kg, p.o. Male Wistar rats were divided into five groups containing twelve animals each. Animals in the sham group (Group I) were pretreated with saline for 4 weeks and underwent the exposure of the left renal artery, but did not receive any occlusion. The animals in RAO group (Group II) received 4 h of RAO and pretreatment of saline for 4 weeks. Groups III, IV and V were pretreatment with naringin (20, 40, and 80 mg/kg) for 4 weeks and then received 4 h of RAO. Out of twelve animals in each group six animals were subjected to hemodynamic measurement and remaining were used for the measurement of left ventricular heart function.

Renal artery occluded renovascular hypertension in rats

After 4 weeks of naringin treatment, animals were anaesthetized by intraperitoneal injection of 1.25 g/kg of Urethane. A small cut was made on the left side of peritoneal cavity of the animal to expose left kidney. The left renal artery was occluded for the 4 h using rat bulldog clamp. The jugular vein was cannulated for the administration of test drug. The right carotid artery was cannulated and connected to the blood pressure transducer of powerlab assembly to measure the hemodynamic changes. After stabilization of blood pressure, the rat bulldog clamp was removed. Then, 1/10th of the administered dose of the naringin, that is, 20, 40 and 80 mg/kg was injected to Group III, IV and V respectively through jugular vein and hemodynamic as well as left ventricular alterations were measured at different time intervals (0, 5, 15, 30, 45, 60 and 90 min).[41]

Invasive measurement of hemodynamic changes

Hemodynamic changes such as heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MABP), left ventricular end diastolic pressure (LVEDP) and Ventricular Contractility Assessment (dp/dt) were measured by means of a polyethylene cannula (PE 50) filled with heparinized saline (100 IU/ml) inserted into the right carotid artery. The cannula was connected to a transducer, and the signal was amplified by means of bioamplifier. Left ventricular systolic pressure was measured by means of a Millar Mikro-Tip transducer catheter (Model SRP-320, Millar Instrument, INC 320-7051, Houston, Texas 77023-5417) inserted into the left ventricle via the right carotid artery and connected to a bioamplifier. The left ventricular functions like dp/dtmax, dp/dtmin and LVEDP signals were obtained from primary signals (left ventricular systolic pressure and blood pressure) by means of an acquisition data system (AD Instruments Pvt. Ltd., with software (LabChart 7.3; AD Instrument Pvt. Ltd.,).[42]

Biochemical estimation

Tissue homogenate preparation

Immediately after assessment of hemodyamic parameters animal were scarified, and kidney tissue homogenates were prepared with 0.1 M tris–HCl buffer (pH 7.4) and supernatant of homogenate was employed to estimate superoxide dismutase (SOD), reduced GSH, lipid peroxidation (malondialdehyde [MDA] content) as described previously.[43]

Histopathological analysis of kidney

Kidney tissue was stored in 10% formalin for 24 h. The specimen was dehydrated and placed in xylene for 1 h (3 times) and later in ethyl alcohol (70, 90 and 100%) for 2 h resp. The infiltration and impregnation were carried out by treating with paraffin wax twice, each time for one h. Tissue specimens were cut into sections of 3-5 μm thickness and were stained with haematoxylin and eosin. The specimen was mounted on the slide by use of distrene pthalate xylene as mounting medium. Sections were examined under a light microscope to obtain a general impression of the histopathology features of specimen and infiltration of cells. The various changes in histological features were graded as Grade 0 (not present or very slight) l; Grade 1 (mild); Grade 2 (moderate); and Grade 3 (severe).

Statistical analysis

Data were expressed as mean ± standard error mean. Data analysis was performed using GraphPad Prism 5.0 software (Graph Pad, San Diego, CA, USA). Data of hemodynamic changes were analyzed using two-way analysis of variance (ANOVA) and Bonferroni's test was applied for post-hoc analysis as well as data of biochemical parameters were analyzed using one-way ANOVA and Dunnett's test was applied for post-hoc analysis. A value of P < 0.05 was considered as statistically significant.

Results

Effect of naringin on heart rate

There was significant (P < 0.001) decreased in HR in RAO control animals from 5, 15, 30, and 45 min as compared to sham animals. Treatment of naringin (40 mg/kg) showed substantial (P < 0.001) improvement in HR at 30 min and 45 min as compared to RAO control rats. Whereas naringin (80 mg/kg) treatment significantly (P < 0.001) increased the HR at 15, 30, 45 and 90 min when compared to RAO control rats [Figure 1a].

Figure 1

Effect of naringin on hemodynamic parameters (a) heart rate, (b) systolic blood pressure, (c) diastolic blood pressure and (d) mean arterial blood pressure in renal artery occlusion (RAO) induced hypertensive rats. Data are expressed as mean ± standard error mean (n = 6) and analyzed by two-way analysis of variance followed by Bonferroni's multiple range test for each parameter separately. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to RAO group and #P < 0.05, ##P < 0.01 and ###P < 0.001 as compared to sham group

Effect of naringin on systolic blood pressure, diastolic blood pressure and mean arterial blood pressure

After occlusion of renal artery, there was conspicuous (P < 0.001) elevation in SBP and DBP of RAO control animals throughout the recording of hemodynamic changes as compared to sham rats. Treatment with naringin (40 mg/kg) showed notably (P < 0.01) alleviation in SBP and DBP at 15, 30, 45, 60 and 90 min as compared to sham rats. On the other hand, naringin (80 mg/kg) treatment showed a significant (P < 0.001) an incredible antihypertensive potential by reinstate SBP and DBP at 15, 30, 45, 60, and 90 min when compared to sham animals. After occlusion of renal artery for 4 h of RAO control rats showed substantial (P < 0.001) increase in MABP as compared to sham rats. On the other hand, treatment with naringin (80 mg/kg) appreciably lessened the MABP at 15, 30, 45, 60 and 90 min when compared to RAO control rats [Figure 1b-d].

Effect of naringin on left ventricular function

Occlusion of renal artery for 4 h resulted in noteworthy (P < 0.001) rise in LEVDP at 30, 45, and 60 min when compared to the sham group. Conversely, treatment with naringin (40 mg/kg) noticeably (P < 0.05 and P < 0.01) ameliorated the LVEDP at 30 min and 45 min as compared to RAO control rats. Administration of naringin (80 mg/kg) illustrated more stupendous antihypertensive effect by significantly lessening (P < 0.001, P < 0.05 and P < 0.001) LVEDP at 30, 45 and 60 min as compared to RAO control rats. There were no imperative changes in maximum first derivative of ventricular pressure (dp/dtmax), and minimum first derivative of ventricular pressure (dp/dtmin) in RAO group after clamp removal and during study period [Table 1].

Table 1

Effect of naringin on left ventricular function in RAO induced hypertensive rats

Effect of naringin on renal superoxide dismutase and glutathione level

Renal SOD and GSH level of RAO control rats were notably decreased (P < 0.001) after occlusion of renal artery for 4 h when compared to sham rats. The renal SOD level in naringin (40 and 80 mg/kg) treated rats was considerably (P < 0.01 and P < 0.001) and dose dependently increased when compared to RAO control rats. Whereas, naringin (40 and 80 mg/kg) treatment showed noteworthy (P < 0.05 and P < 0.001) and dose dependent increased in the GSH level when compared to RAO control animals [Table 2].

Table 2

Effect of naringin on oxidative stress in RAO induced hypertensive rats

Effect of naringin on renal malondialdehyde level

After occlusion of renal artery, lipid peroxidation (MDA) level in kidney was considerably increased (P < 0.001) in RAO control rats when compared to sham rats. The renal MDA level in naringin (40 and 80 mg/kg) treated rats was lessened appreciably (P < 0.01 and P < 0.001) and dose-dependently when compared to RAO control rats [Table 2].

Effect of naringin on histopathology of kidney

Histopathological analysis of kidney from sham control rats showed normal architecture of the kidney. It was devoid of congestion, necrosis and inflammatory infiltration (Grade 0) [Figure 2a]. Basement membrane of the glomerulus was remained intact. Kidney form RAO control rats showed renal damage evident by glomerular structural disruption. It showed the presence of intraluminal cell debris, edema and inflammatory infiltration [Figure 2b]. Histological analysis of kidney from naringin (40 and 80 mg/kg) treated rats showed moderate number of inflammatory cells without any necrosis and edema. Thickens of glomerulus basement membrane was intact [Figure 2c, d and Table 3].

Figure 2

Effect of naringin on histopathology alteration produced by renal artery occlusion (RAO) in rat kidney. Photomicrograph of sections of kidney of (a) normal, (b) RAO control rats, (c) naringin (40 mg/kg) treated rats and (d) naringin (80 mg/kg) treated rats. Necrosis (black arrow), inflammatory infiltration (red arrow) and increased thickness of basement membrane (yellow arrow) H and E staining at × 100

Table 3

Effect of naringin on kidney histopathology in RAO induced hypertensive rats

Discussion

Renovascular hypertension is a syndrome that consists of high blood pressure caused by the kidneys hormonal reaction to tapering of the arteries supplying the kidneys. RVHT reflects the fundamental relation between anatomically manifest arterial occlusive ailment and eminent blood pressure.[44] ROA produces ischemia, which instigates the release of renin and a secondary altitude in blood pressure. Rennin endorses conversion of angiotensin-I to angiotensin-II, causing severe vasoconstriction and aldosterone release. RVHT emerges in due to RAO, habitually from atherosclerotic or fibromuscular dysplastic renal disease, since this worsening of renal perfusion pressure causes the kidney to engender surfeit of renin and leads to a persistent commencement of the rennin-angiotensin-aldosterone system.

The present study assessed isolated bioactive moiety naringin to protect against renovscular hypertension in laboratory animals. The literature is scattered with affirmations indicating antioxidants, antiapoptotic, antiinflammatory and ROS scavenging properties of naringin, as the prime components of renovascular damage is generation of ROS leading to vascular damage hence the selection of naringin in this animal model is astute.

The present study of RAO depicts that clamping of renal artery for 45 min. may prompt the RAO induced oxidant damage and treatment of naringin may supersede the defense that was afforded by restitution of hemodynamic alterations in the kidney. Allied with the hypertension of renal artery clipping, the myocardium endures a hypertrophic response as variation to amplified blood pressure.[45] The most liable enlightenment for our findings is that pretreatment with naringin appreciably lessen hemodynamic parameters such as systolic, diastolic, MABP and HR. The utmost boost in systolic, diastolic, MABP and end-diastolic pressure in RAO group was observed at 15 min. after clamp deletion, after which the blood pressures leaned to recuperate up to 60 min. Treatment of naringin illustrated the momentous drop in systolic, diastolic, MABP and end-diastolic pressure at 15 min after clamp removal, and radically improved up to 60 min. Due to clamping on renal artery inconsequential varies were scrutinized in maximum first derivative of ventricular pressure (dp/dtmax), minimum first derivative of ventricular pressure (dp/dtmin).

An assortment of enzymes is conscientious for the cohort of ROS including nitric oxide synthase, NADH/NADPH oxidase, cyclooxygenase, and xanthine oxidase; the mitochondrial electron transport chain is also involved in the generation of ROS.[464546474849] Oxidative stress has a superfluous of components that encompass an assortment of enzymes implicated in the electron transport chain. Moreover, oxidative stress can elicit inflammation, enhance vasoconstrictors accrual, and endorse tubular cell apoptosis and necrosis.[5051] This higher level of ROS diminishes the copper-zinc-SOD enzyme motion alongside GSH performance in cardiac cells, which causes consequential hammering of myocardial integrity and necrosis.[525354] The prominent cause of lipid peroxidation is an excessive generation of ROS by superoxide anions and their derivatives including hydroxyl radicals in cardiac myocytes.[55] Furthermore, through adaptation of cellular protein and DNA damage lipid peroxidation is measured as foremost cause of oxidative damage.[1756] RIR injury is pigeonholed by a surfeit of ROS formation and a diminutive of antioxidants, resulting in mounting oxidative stress and tissue damage.[457] Concrete with this notion, we found that renal IRI drastically lessened SOD, GSH levels and elevated MDA content. This augmented oxidative stress was allied with prejudiced renal function and histological changes as noticed by idiosyncratic renal morphological variations. Conversely, bioflavonoid naringin prohibited severe tiredness of SOD activity in depressed kidney and restrained IRI induced lipid peroxidation and subsequently enhanced kidney function and dent caused by RAO. Research carried out by various workers showed that naringin exerts its protective role via regulating antioxidative capacity by increasing SOD activity and by up-regulating the gene expression of SOD and GSH in renal as well as other diseases.[34394058] Results of the present investigation are in line with the findings of these researchers. To sum up, naringin exerts antihypertensive potential via its antioxidant activity. However, further study is undertaken to evaluate the exact mechanism of action of naringin in renal artery occluded RVHT.