JMBD

Introduction

Kidney is an important organ, responsible of the elimination of blood soluble wastes and sustainability of water and electrolyte balances in the body. Unfortunately, 20 to 40% of diabetic mellitus (DM) patients develop diabetic nephropathy (DN) during their lifetime [1]. Among the various and complex mechanisms implicated in DN complications, the conditions such as: the persistent hyperglycaemia-inducing polyol and hexosamine pathways activation, the excess/inappropriate activation of protein kinase C (PKC) isoforms, the increase in reactive oxygen species (ROS), the advanced glycated end-products (AGEs) and pro-inflammatory cytokines production are the major causative and development factors [2,3]. Studies reported that kidney oxidative stress and chronic inflammation contribute equally to the development and progression of DN [4]. The increase oxidative stress results in imbalance of mitochondrial redox activities, followed by the activation of caspases and depletion of cellular antioxidant enzymes to promote further excessive reactive oxygen species (ROS) formation, DNA fragmentation and cellular apoptosis [2,5]. The inflammatory processes revealed in DN pathogenesis are NF-kB activation-based cascade reactions [6] with monocytes infiltration and injuries in the glomeruli and tubules [7]. Consequently, inflammatory response, thickening of basement membranes, expansion of mesangial matrix and interstitial fibrosis, hypertrophy and glomerular epithelial cell loss occur and result in increased albuminuria and renal dysfunction [8,9]. Clinically, increased albuminuria and decreased metabolic wastes elimination are good indexes of DN [10]. The treatment protocol of the disease includes predominantly diabetes control and lowering high blood pressure. Indeed, studies described angiotensin converting enzyme inhibitors (ACEIs) agents as adequate and effective treatment against DN progression [11]. However, health workers encounter difficulties to manage successfully DN due to the complexity of the involved mechanisms and the absence of early symptoms. Hence, in the absence of reliable and effective allopathic drug therapies, plants remedies are being extensively used as alternative medicines for better control and management of diabetic nephropathy [12]. Togo and Sub-Saharan Africa as well as other parts of the world have very rich history of herbal medicine used since decades against ailments in human. Studies have confirmed that secondary metabolites of plants are diversified low molecular weight compounds with wide range of biological properties through interaction with proteins, nucleic acids and various membrane receptors [13]. Therefore, plant derivatives are increasingly getting attention in drug development domains as to date; more than 50 % of all drugs in clinical use are either natural products or their derivatives [14].

More studies need to be carried out to improve medicinal plant’s use in our communities where a plant remedy can be used to treat a broad spectrum of diseases. Indeed, the seeds of Nigella sativa (Ranunculaceae) is traditionally used as an anti-inflammatory, antioxidant, anticancer, nephroprotective, hepathoprotective, antidiabetic, anti-ulcer, antiseptic, immunomodulatory and analgesic remedy [15]. In addition, the leaf extract of Ginkgo biloba (Ginkgoaceae) known for its antioxidant activities relying mainely on bilobalide (terpenoids), quercetin and kaempferol (flavonoids) are used in the management of cancer, neurodegenerative and cardiovascular disease [16]. In order to develop phytopharmaceuticals to improve human healthcare, many studies have evaluated and confirmed the antioxidants properties of some common plants used as home therapy or food. This include studies on Amaranthus hybridus (Linn.) (Amaranthaceae) leaf, seed or whole plant used for its anti-viral, anti-bacterial, anti-fungal and anti-inflammatory properties; Allium sativum L. (Amaryllidaceae) bulb or whole plant used in the treatment of atheroclerosis, hyperlipidemia, hypertension, diabetes mellitus, bacterial infections, cancer, fever, dyspepsia, intestinal worms and tuberculosis; Vernonia amygdalina ( Asteraceae ) leaves used to treat dysentery, gastrointestinal tract disorders [17]. The use of bamboo leaf is very common in Chinese, Indian and Japanese food as well as in both traditional and modern medicine, and investigations on bamboo species for other applications such as food additives have been documented  [18,19]. In Sub-Saharan Africa where O. abyssinica (Poaceae), a bamboo species is endemic, the leaves of the plant are being used as remedy against DN symptoms mainly polyurea, albuminuria [19]. Early studies mentioned that extracts of bamboo leaf could ameliorate cell apoptosis mechanisms associated with DN pathogenesis [20]. Meanwhile, though extracts of O. abyssinica have been documented for their antioxidant capacity and antidiabetic activities [21,22], no study has yet investigated their possible effects in DN. The protective effects of methanol extract and fraction of O. abyssinica against renal oxidative stress and dysfunction are presented in this study.

Materials and Methods

Chemicals and drug

Alloxan monohydrate, methanol, ethyl-acetate, n-hexane and ethyl ether (Sigma Aldrich Chemical Co., St. Louis, MO, USA), D-glucose and Sodium Chloride (BDH/Merck, Poole, UK); Vasotec (Enalapril, Merck & Co. UK), Accu-check glucometer active (Roche Diagnostics Co., Mannheim, Germany), Distilled water (ACEPRD, University of Jos, Jos, Nigeria), Ellman reagents and TBA (BDH Laboratory supplies, Poole Dorset BH 15 1TD, UK); hydrogen peroxide (DE-SHALOM Pharm. Lab. Ltd OkeOye Osun State, Nigeria); GSH (Loba Chemie Wodehouse Road, Mumbai, India).

Plant material

O. abyssinica fresh leaves were collected from Kpéwa, Togo in December 2018 and authenticated by Professor Atsu K. GUELLY in Botany and Plant Ecology Laboratory, Faculty of Sciences, University of Lomé, Togo. A voucher specimen of Herbarium accession No: TOGO15189, was deposited in the herbarium of the Department of Botany. The leaves were shade dried and then powdered. Extract and fractions preparation The extraction was carried out with 500 g of the powder soaked in 2.5 L of 80% methanol in a glass jar. The mixture was macerated under periodic agitation for 72h at room temperature and filtrated. A direct exhaustive and successive fractionation was carried out on another 500 g of the powder using solvents with increase polarity (n-hexane, ethyl acetate and methanol). The filtrates were concentrated under reduced pressure in the Buchi rotavapor R-200. The methanol extract (Met-E) and methanol Fraction (Met-F) were collected and stored in a glass container at -4°C till further use.

Animal material

Animals Male Albino Wistar rats of 4 weeks age were purchased from animal experimental unit of University of Jos. They were put into clear and dry cages and housed at room temperature with 12 h light/dark cycle in the same animal experimental unit where they were allowed free access to laboratory standard chow and tap water for 6 weeks. The rats weighing between 170-200 g were selected and used in the experiments.

Experimental design

Diabetic was induced by single intraperitoneal injection of 150 mg/kg body weight (b.w.) of alloxan monohydrate (ALX) freshly prepared in normal saline. After 48 h, the serum glucose (SGlu) level of the rats was checked using Accu-Check Active glucometer (Roche, Germany). Rats with SGlu level ≥200 mg/dL were selected as diabetics and included in the study when they were still confirmed diabetics 4 weeks after the injection. For the experiment, animals were separated into metabolic cages which were numbered according to the grouping method and each group comprised of 3 rats. The Non-diabetic control or Normoglycaemic control (NGC) group (G1) and the Diabetic control (DC) group (G2) were orally given distilled water while groups: G3, G4 and

G5 were orally given once daily Met-E (400 mg/Kg b.w.), Met-F (400 mg/kg b.w.) and Enalapril (10 mg/kg b.w.) respectively for 27 days. At the end of the experiment on the 28th day, 24 hours urine was collected through the metabolic cage system and blood samples were collected from jugular vein immediately after the rats were euthanized by decapitation under 40% ethyl ether anaesthesia to avoid stress condition. Both kidneys were removed. The right kidney tissues were rinsed thoroughly with normal saline then weighed, minced and a homogenate was prepared with 1 g/ 4 mL (w/v) of the tissue in another phosphate-buffered saline (0.1 mol/L, pH 7.4) to get proteins extracted. The homogenate was separated into two portions and the first portion was used to estimate: Malondialdehyde (MDA) and reduced glutathione (GSH) levels. The second portion was centrifuged (6000g; - 4°C) for 20 min to obtain post-mitochondrial supernatant which was used to estimate superoxide dismutase (SOD) and catalase (CAT) activities. The left kidney tissues were fixed in 10 % formalin for histopathological investigations.

Serum Glucose and renal function biomarkers

Serum glucose (SGlu), serum creatinine (SCr), urine albumin (Ualb) and blood urea nitrogen (BUN) from serum urea were determined using the automatic Cobas c111 biochemistry analyser (Roche) in the biochemistry laboratory of Plateau Special Hospital, Jos, Plateau State.

Estimation of MDA products level

Lipid peroxidation was assessed in terms of MDA formation, according to the method of  Varshney and Kale [23]. The absorbance was measured against blank at 532 nm. MDA formed (mmol/mg protein) was calculated using the molar extinction coefficient of

1.56x10⁵M^-1Cm^-1. 

Renal antioxidants estimation

GSH level was estimated by the method of Beutler and Yeh [24], CAT activity measured through the breakdown of hydrogen peroxide was determined according to Sani et al. [25] method. The SOD activity was estimated based on the ability of the enzyme to inhibit the auto-oxidation of epinephrine at pH 10.2 using the method of Misra and Fridovich [26]. The unit of SOD activity is defined as the enzyme required for 50% inhibition of adrenaline auto-oxidation. Renal protein contents estimation The total protein content in the kidney was estimated by means of Biuret method as described by Gornall et al. [27]. Histopathological investigations The renal histopathology study was assessed to evaluate the oral administration effect of the Met-E and the Met-Fon the renal tissues of 8 weeks diabetic rats. The study was done based on the methods previously described [28,29]. Tissues were processed, paraffin embedded and sections were stained in Harris’ haematoxylin and counterstained in 1% aqueous eosin for examination under light microscope.

Renal protein contents estimation

The total protein content in the kidney was estimated by means of Biuret method as described by Gornall et al. [27].

Histopathological investigations

The renal histopathology study was assessed to evaluate the oral administration effect of the Met-E and the Met-F on the renal tissues of 8 weeks diabetic rats. The study was done based on the methods previously described [28,29]. Tissues were processed, paraffin embedded and sections were stained in Harris’ haematoxylin and counterstained in 1% aqueous eosin for examination under light microscope.

Statistical data analysis

GraphPad Prism software version 8.0.1 (San Diego, California USA) was used for data analysis. Difference between groups were analysed using one-way analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons. The results are expressed as mean ± SEM (n=3) and difference between groups were considered significant at p<0.05.

Results

Serum glucose and renal function biomarkers

The effect of the treatments on the evaluated renal function parameters is summarised in table 1. The DC rats showed a significant (p<0.001) increase of 271.91% (19.86±1.59 mmol/L) in SGlu level versus NGC rats (5.34±0.37 mmol/L). Four weeks administration of Met-E and Met-F to diabetic rats resulted in a significant (p<0.05) decrease in SGlu: 54.23% (9.09±2.39 mmol/L) and 55.94% (8.75±2.95 mmol/L) respectively. The

results in Enalapril-treated rats revealed a non-significant (p>0.05) decrease of SGlu level (14.39±2.48 mmol/L) as compared to DC (19.86±1.59 mmol/L). A Significant (p<0.0001) increase of 156.18% (12.86±1.36 g/L) in Ualb level was observed in DC versus values in NGC group (5.02±0.58 g/L). A Significant (p<0.0001) decrease in Ualb level was recorded with Met-E: 74.96% (3.22±0.13 g/L), Met-F: 72.94% (3.48±0.12 g/L) and Enalapril: 68.90% (4±0.29 g/L) after administration versus diabetic control (12.86±1.36g/L). The DC rats showed a significant (p<0.0001) increase of 197.48% (46.08±0.4 g/L) in BUN level versus values in NGC rats (15.49±1.3 g/L). Administration of Met-E, Met-F and Enalapril resulted in a slight and non-significant (p>0.05) decrease in BUN level versus DC rats. A slight and non-significant (p>0.05) increase in SCr level was observed in DC animals and no decrease effect was exerted after Met-E or Met-F administration. Nevertheless, Enalapril treated group showed a slight decrease in SCr level which was not significant (p>0.05).

Table 1: Effect of treatments on UAlb, SCr, BUN and SGu after 28 days treatment

The values are expressed as mean ± SEM (n=3). ^ap<0.001; ^bp<0.0001 (Diabetic Control vs Non-Diabetic Control). *p<0.05 and ***p<0.0001 (Treated vs Diabetic Control).

Effect of treatment on MDA products formation

Alloxan-induced diabetic rats showed a significant (p<0.0001) increase of 232.56% (1.43±0.09 mmol/mg protein) in MDA level compared to NGC rats. The administration of Met-E exhibited a significant (p<0.001) decrease of 50.35% (0.71±0.12 mmol/mg protein) in MDA level compared to DC rats while the decrease observed in Met-F was not statistically significant. The administration of Enalapril also showed a significant (p<0.001) decrease of 53.15% (0.67±0.10 mmol/mg protein) in MDA level when compared to DC rats (Figure 1).

Figure 1: Effect of MET-E and MET-F on MDA level. The values are expressed as mean ± SEM (n=3). $p<0.0001 (Diabetic Control vs Normoglycaemic); ***p<0.001 (Treated vs Diabetic control).

Effect of treatment on SOD activity

A significant (p<0.05) increase of 114.81% (1.16± 0.25 unit/mg protein) in SOD activity was observed in diabetic untreated rats compared to NGC rats. Administration of Met-E, Met-F and Enalapril exhibited a significant (p<0.05) decrease of 50.86% (0.57±0.09 unit/mg protein), 56.90% (0.50±0.04 unit/mg protein) and 62.93% (0.43±0.11 unit/mg protein) respectively in SOD activity when compared to Diabetic Control rats (Figure 2).

Figure 2: Effect of treatment on SOD activity. The values are expressed as mean ± SEM (n=3). *p<0.05 (Diabetic control vs Normoglycaemic and Treated vs Diabetic control).

Effect of treatment on GSH level

GSH values were significantly (p<0.0001) decreased by 68.52% (15.59±1.73) in diabetic untreated rats compared to NDC rats.

GSH activity was significantly (p<0.05) increased by 85.18% (28.87±1.81) in diabetic rats after Met-E Administration. The administration of Met-F and Enalapril caused a significant (p<0.001) increase of 123.22% (34.80±3.0) and 143.43% (37.95±4.55) respectively compared to diabetic control rats (Figure 3).

Figure 3: Effect of treatment on GSH level. The values are expressed as mean ± SEM (n=3). $p<0.0001 (Diabetic control vs Normoglycaemic); *p<0.05 and **p<0.01 (Treated vs Diabetic control).

CAT activity

The diabetic untreated rats showed significant (p<0.001) decrease of 53.95% (189.07±9.02 µg/ml) in CAT activity compared to NGC. The administration of Met-E showed a significant (p<0.001) increase of 134.95% (444.22±39.66 µg/ml) in CAT activity compared to diabetic control. No significant increase was observed in Met-F and Enalapril-treated rats (Figure 4).

Figure 4: Effect of treatment on CAT activity. The values are expressed as mean ± SEM (n=3). ***p<0.001 (Diabetic control vs Normoglycaemic and treated vs Diabetic Control).

Histopathology Assessment

The effect of treatment on the histology of the kidney of the experimental rats was shown in (Figure 5). The kidney section from normal rat presented normal morphology with intact cell nuclei (white arrowheads) surrounded by intact cytoplasmic content. The glomeruli (white stars) were numerous and clearly surrounded by a capsular space (black arrowheads). The glomerular capsules (black arrows) were also intact with their characteristic simple squamous epithelial cells. The convoluted tubules (white arrows) and their epithelial lining were normal (A0). ALX has induced a significant renal damage evidenced in diabetic untreated (DC) rats by loss of tissue components and architecture (black arrows) with severe glomerular congestion and degeneration, severe inflammatory cells (white stars) infiltration and necrosis (A1). A section of the kidney of diabetic rats treated with Met-E showed the presence of glomeruli (white stars) with reduced capsular space as a result of hypertrophy and minor inflammatory cells (white arrows) within the tissue compared to DC. Tissue exhibited also the presence of few red blood cells (white arrowheads) and clear presence of tubules (black arrows) (A2). A Section of the kidney of diabetic rats treated with Met-F showed tissue degeneration with massive loss of tissue components. However, minor infiltration of inflammatory cells (black arrows) was displayed compared to DC and some necrosis spaces remained empty(black arrowheads). The glomeruli (white stars) are hypertrophied and congested with red blood cells (white arrows) (A3). A Section of the kidney of diabetic rats treated with Enalapril showed tissue degeneration with massive loss of tubular morphology (White arrows). Infiltration of inflammatory cells (white arrowheads) also appeared within the tissue but minor than DC. Interspersing the necrotic tissue were normal cells presented with normal nuclei (black arrows) surrounded by intact cytoplasmic components (A4).

© by the Authors & Gavin Publishers. This is an Open Access Journal Article Published Under Attribution-Share Alike CC BY-SA: Creative Commons Attribution-Share Alike 4.0 International License. Read More About Open Access Policy.