Animals
Thirty-six male Albino Wistar rats weighing 180–220 g were used for this experimental examination. Rats were procured from animal house facility of the R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist-Dhule-Maharashtra). Animals were placed in nicely ventilated polypropylene cages and maintained under an ambient temperature of 25 ± 2 °C, 12-h light/dark cycle in the departmental animal house. The animals had been fed with trendy pelletized feed (Amrut Rat Feed, Pune) and water ad libitum. The experimental protocol was pre-accepted by the institutional animal ethical committee (IAEC), protocol approval no. RCPIPER/IAEC/2018-19/06.
Chemicals and drugs
STZ, formononetin, metformin, and carboxymethylcellulose were obtained from Sigma Aldrich (Mumbai, India). Glucose kit, creatinine kit, urea/BUN kit, albumin kit, and total protein kit were received from ERBA (Mumbai, India). Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were purchased from Loba Chemicals Pvt Ltd (Mumbai, India). All other chemicals used in the study were of analytical grade.
Induction of diabetic nephropathy
Diabetes was induced in rats by a single dose of STZ (55 mg/kg) intraperitoneal route except for the normal group. STZ was prepared in cold phosphate buffer solution (pH-7.4) in dark conditions. After 72 h of STZ administration, DN was induced in animals [13].
Experimental design
Rats were randomly divided into six groups (n = 6).
Group 1: Normal - received 0.5% CMC
Group 2: Diabetic nephropathic group (DN) - injected with STZ (55 mg/kg)
Group 3: DN + Standard drug (Metformin) - injected with STZ (55 mg/kg) + metformin (70 mg/kg)
Group 4 to 6: Injected with STZ (55 mg/kg) and 10, 20, and 40 mg/kg of formononetin, respectively.
Metformin and formononetin suspension was prepared in 0.5% CMC and administered by oral route for 14 days. On the 15th day, animals were anesthetized by urethane (1.4 mg/kg) [14] and blood samples were collected. For separation of the serum, the blood was centrifuged at 5000 rpm for 10 min at 4 °C and the serum was separated and used for further biochemical estimation. Finally, animals were sacrificed and both kidneys were isolated. A 10% tissue homogenate of the kidney was prepared using ice-cold 50 mM phosphate buffer saline (pH 7.4). The homogenate was centrifuged at 10000 rpm for 10 min at 4 °C, and the supernatant was separated and used for further estimation.
Physiological parameters
The body weight of each animal was recorded weekly by using an electronic weighing balance. At the end of the experiment, the weight of both kidney and liver was taken.
Assessment of kidney function
On the 14th day of study, rats were kept individually in metabolic cages for 24 h urine collection for the measurement of urine output and renal function. Before urine collection, blood samples were collected by retro-orbital puncture for separation of the serum. Renal function was assessed by measuring serum levels of fasting glucose by the glucose oxidase-peroxidase method, creatinine, and BUN, and blood albumin was measured using the commercially available marketed kit (Erba kit) [15].
Biochemical estimations in kidney homogenates
Renal oxidative stress biomarkers like GSH, SOD, and MDA were estimated using commercially available marketed kits according to the manufacturer’s guidelines, and CAT was estimated using commercially available UV spectroscopic methods. Briefly, the kidney homogenate supernatant (10 μL) was added to 0.5 mL of 10 mM hydrogen peroxide (H2O2) solution. The reduction in optical density of this mixture was measured at 240 nm by using a UV spectrophotometer. The rate of decrease in the optical density within 3 min from the addition of kidney homogenate was taken as an indicator of the catalase activity present in the homogenate [16].
Cytokine parameters
Proinflammatory cytokines like TNF-α and IL-6 were measured in kidney homogenate by using respective kits according to the manufacturer’s protocol, and the final concentration was determined by using the standard curve [17].
Histopathological examinations
The kidney was removed from each rat by injecting a high dose of anesthesia (urethane 1.4 mg/kg i.p.) and buffered in formalin (10%) solution and embedded in paraffin wax. The sample was then cut into around 5-μm-thick sections with a microtome and deparaffined with xylene. Then, sections were subjected to standard staining of hematoxylin/eosin. Sections were observed under a light microscope at magnifications of ×40. Light microscopy was used to evaluate tubular necrosis and glomerular damage [18].
Statistical analysis
Data were presented as mean ± S.E.M. of each group. The result values were statistically analyzed by using one-way ANOVA and two-way ANOVA followed by Bonferroni’s test using graph pad prism version 7.0. A difference was regarded as significant when P < 0.05, P < 0.01, and P < 0.001.
