Crude extract extraction
Momordica balsamina (MB) was identified by a botanist at the University of KwaZulu Natal, Westville. The plant was harvested at the University of KwaZulu Natal in Westville. The leaves were washed three times with water to remove any residual dirt.
Extraction Method
Methanolic extract (ME) was extracted from MB leaves by using a standard protocol that has been validated in our laboratory with minor modifications (18). Briefly, the air-dried MB leaves were sequentially extracted twice at 24 h intervals at room temperature using methanol (45 mL) and deionised water (45 mL) on each occasion. The solvent was removed from the crude extract under reduced pressure at 55 ± 1 ºC using rotatory evaporator to yield dichloromethane solubles (DCMS) and ethyl acetate soluble (EAS).
Animals
Thirty-six (36) male Sprague Dawley rats with body weight 150–180g were used for this study. The rats were obtained from the Biomedical Research Unit (BRU), University of KwaZulu-Natal (UKZN). The animals were kept and maintained in a standard animal facility under controlled environmental conditions at room temperature (22 ± 2°C), humidity (55 ± 5%) and 12h day:12h night cycle. The animals consumed standard rat chow (Meadow Feeds, South Africa) and water ad libitum for 2 weeks to acclimatize before being exposed to the experimental diet (high–fat high–carbohydrate). The components of the high–fat high–carbohydrate (HFHC) diet are carbohydrate (55% Kcal/g), fats (30% Kcal/g) and proteins (15% Kcal/g). All experimental procedures in this study were carried out in absolute compliance with the animal ethics and animal guidelines of the Animal Research Ethics Committee (AREC, ethics no: AREC/062/018M) of the UKZN, Durban, South Africa.
Induction Of Prediabetes
After acclimatization, the animals were divided into 2 main groups: the non-prediabetic control group (n = 6) and the prediabetic group (n = 30). The normal control (NC) animals (negative control) were given normal diet (ND) and water ad libitum for 20 weeks while the prediabetic animals were given HFHC diet and drinking water supplemented with fructose (15%) for 20 weeks to induce prediabetes. At 20th week, prediabetes was confirmed via assessment of fasting blood glucose concentration and oral glucose tolerance test (OGTT) as described by (19).
Experimental Protocol
After prediabetes induction, the non-prediabetic control group (Group 1) continuously fed on ND and received diluted dimethyl sulphoxide, DMSO (2 ml DMSO: 19 ml normal saline) as a vehicle for 12 weeks while the prediabetic animals (n = 30) were further divided into 5 groups (n = 6). All the prediabetic animals continuously fed on either HFHC or ND and were treated with either oral administration of MB (250mg/kg) or metformin (MET, 500mg/kg) every third day for 12 weeks. The prediabetes control group, PD (Group 2) rats were continuously fed on the HFHC diet and received the diluted DMSO (vehicle) for 12 weeks. The ND + MET (Group 3) rats changed the diet to ND and received MET orally, whereas the HFHC + MET (Group 4) rats were continuously fed on the HFHC diet and received MET orally. The ND + MB (Group 5) rats changed the diet to ND and received MB orally while HFHC + MB (Group 6) rats continuously fed on the HFHC diet and were treated with MB. The fluid intake and urine volumes were assessed in all the animals at 20th week and every 4 weeks (24th, 28th and 32nd week). These therefore means the animals were place at metabolic cages on the day fluid intake and urinary output measurement. After the 12 weeks of treatment, the animals were sacrificed, blood samples and the kidneys were collected from all the animals for biochemical analysis.
Blood Collection And Tissue Harvesting
All the animals were placed in a gas anaesthetic chamber (Biomedical Research Unit, UKZN, Durban, South Africa) and anaesthetised with 100 mg/kg of Isofor (Safeline Pharmaceuticals (Pty) Ltd, Roodeport, South Africa). In an unconscious state, blood samples were collected from all the animals via cardiac puncture into different pre-cooled EDTA containers. The blood samples were centrifuged (Eppendorf centrifuge 5403, Germany), 503 g for 15 minutes at 4°C to obtain plasma. Thereafter, the plasma samples were aspirated into plain sample bottles and stored in a Bio Ultra freezer (Snijders Scientific, Tilburg, Holland) at -80°C until ready for biochemical analysis. Also, the kidneys were removed, rinsed with cold normal saline solution, weighed on the weighing balance, snapped frozen in liquid nitrogen and stored at -80°C in a Bio Ultra freezer for biochemical analysis of selected parameters.
Biochemical Analysis
The biochemical analysis of kidney function parameters (such as creatinine, urea, uric acid, albumin and total protein) and electrolytes (Na+ and K+) were determined at 32nd week in the plasma and urine samples by using their respective assay kits (Elabscience Biotechnology Co., Ltd., Houston, TX, USA) as instructed by the manufacturer. The kidney injury molecule (KIM-1) and plasma aldosterone concentrations were determined using respective specific ELISA kits as instructed by the manufacturer (Elabscience Biotechnology Co., Ltd., Houston, TX, USA) via the microplate reader, Spectrostar Nano spectrophotometer (BMG Labtech, Ortenburg, LGBW, Germany)
Lipid Peroxidation And Antioxidant Status (Dup: Abstract ?)
The lipid peroxidation was assessed by determination of the concentration of malondialdehyde (MDA) in the kidney homogenized tissue according to the previously established protocol (Mkhwanazi et al. 2014). The antioxidant status of the kidney homogenate was assessed by determination of the concentration of superoxide dismutase (SOD), glutathione peroxidase (GPx) and total antioxidant capacity (TOAC) by using their specific assay kits according to the instruction of the manufacturer (Elabscience Biotechnology Co., Ltd., Houston, TX, USA).
Urine Rna Isolation
RNA was isolated from urine (4 ml) by using ZR Urine RNA Isolation KitTM (Zymo Research Corp, Irvine, USA) according to the manufacturer’s protocol. The purity of the RNA was confirmed by the relative absorbance of ratio 260/280 nm via Nanodrop 1000 spectrophotometer (Thermo Scientific, USA). Urine RNA (100 ng) was reverse transcribed to complementary DNA (cDNA) by using iScriptTM cDNA Synthesis Kit (Bio Rad, California, USA) through incubation in a thermal cycler (SimpliAmp Thermal Cycler, Applied biosystems, Life technologies).
Urine Complementary Dna (Cdna) Synthesis
For cDNA synthesis, urine RNA (2 µL) was mixed with 5X iScript reaction (4 µL), iScript reverse transcriptase enzyme (1µL) (Bio Rad, USA) and nuclease-free water to a final volume of 20 µL. The mixture was incubated in the thermal cycler (SimpliAmp Thermal Cycler, Applied biosystems, Life technologies) at 25oC for 5 minutes, 42oC for 30 minutes and finally at 85oC for 5 minutes. Thereafter, the synthesized cDNA was stored at -80°C until use for real-time PCR (Polymerase chain reaction).
Real-time Pcr
The urinary mRNA level of podocin was quantified by real-time PCR lightcycler (Roche LightCycler 96, USA). cDNA template (2 µL), SYBR Green PCR master mix (5 µL) (Bio Rad, USA), podocin forward primer (1 µL), podocin reverse primer (1 µL) and nuclease-free water were mixed to a final volume of 10 µL. Thereafter, the sample mixtures were cycled 40 times at 95oC for 10 seconds, 60oC for 20 seconds and 72oC for 20 seconds in the lightcycler (Roche LightCycler 96, USA). All the samples were run in duplicate, and β-actin mRNA levels were used as a housekeeping gene to normalize the podocin mRNA level. The sequences of the used oligonucleotide primers (Metabion International AG, Planegg, Germany) were as followed: podocin forward 5`-TGG AAG CTG AGG CAC AAA GA-3`, podocin reverse 5`-AGA ATC TCA GCC GCC ATC CT-3`.
Statistical analysis
The data were presented in mean ± SEM and analysed via a two-way Analysis of Variance (ANOVA) with the Bonferroni test as a post hoc test by using GraphPad Prism 5 software. The results were considered to be statistically significant from p < 0.05 and above.