Sodium fluoride, L- Arginine , xylenol orange (XO), potassium hydroxide, reduced glutathione (GSH), oxidized glutathione (GSSG), Sodium fluoride, thiobarbituric acid (TBA), trichloro acetic acid (TCA), sodium hydroxide, O‑dianisidine, and hydrogen peroxide (H2O2), 1,2‑dichloro‑4‑nitrobenzene, were purchased from Sigma (St. Louis, MO, USA). Normal goat serum, Biotinylated 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System with DAB solution were purchased from Elabscience Biotechnology®, China), anti- Angiotensin Converting Enzyme1 Polyclonal Antibody (E-AB-16159: 1:500 Dilution) and Anti-mineralocorticoid receptor Polyclonal Antibody (E-AB-70261: 1:50 Dilution). All other chemicals used for this study were of analytical grade.
Experimental Animals and Design
Thirty male Wistar rats (150 – 180 g) were used in this study, the rats were randomly divided into five groups of six rats each as Control, NaF (300 ppm), NaF + L- Arginine (100 mg/kg), NaF + L- Arginine (200 mg/kg), and NaF + Lisinopril (10 mg/kg), respectively; orally for eight days. The concentration of NaF (Oyagabemi et al. 2021) and the dosages of L- Arginine (Saad 2021) and Lisinopil (Oyagabemi et al., 2021) were chosen based on the previous literature. The animals were also fed with rat cubes ad libitum and water was supplied liberally. The rats were kept in wire mesh cages under controlled light cycle (12 h light/12 h dark) and fed with commercial rat chow ad libitum and liberally supplied with water. The blood of the rats was taken on the 8th day and rats were sacrificed on the 9th day.
The study was conducted following guidelines approved by the Animal Care and Use Research Ethics Committee (ACUREC) of the University of Ibadan (Approval number: UIACUREC/ 19/124).
Blood pressure measurement
The systolic (SBP), diastolic (DBP), and mean arterial (MAP) blood pressures were determined non-invasively in conscious animals by tail plethysmography using an automated blood pressure monitor (CODA S1, Kent Scientiﬁc Corporation, Connecticut, USA). The blood pressure parameters were obtained by an indirect method of blood pressure measurement as recently reported from our laboratory (Oyagbemi et al. 2019).
The serum was obtained from whole blood collected into anticoagulant free sample bottles following a post-collection waiting period of 60 mins. Thereafter, the serum was kept at a 4oC temperature.
Determination of serum markers of renal damage
Serum creatinine and blood urea nitrogen (BUN) were determined following the manufacturer’s instructions in the purchased Randox® kits (Randox® Laboratories Ardmore, United Kingdom).
Preparation of renal post mitochondrial fractions
The kidney and testes were quickly excised, rinsed, weighed and homogenized with homogenizing buffer (0.1M phosphate buffer, pH 7.4) using a Teflon homogenizer. The homogenate was centrifuged at 10,000 g for 10 minutes at -4OC.
Estimation of renal oxidative stress
Hydrogen peroxide generation was determined according to the method of Wolff (1994). The reaction mixture was subsequently incubated at room temperature for 30 minutes. The mixtures were read at absorbance of 560 nm and H2O2 generated was extrapolated from H2O2 standard curve. The Malondialdehyde (MDA) content as an index of lipid peroxidation was quantified in the PMFs of cardiac and renal tissues according to the method of Varshney and Kale (1990). The absorbance was measured against a blank of distilled water at 532 nm. Lipid peroxidation was calculated with a molar extinction coefficient of 1.56 × 105/M/cm. Protein carbonyl (PCO) contents in the renal and cardiac tissues were measured using the method of Reznick and Packer (1994). The absorbance of the sample was measured at 370 nm. The carbonyl content was calculated based on the molar extinction coefficient of DNPH (2.2 104 cm1 M1) and expressed as nmoles/mg protein while vitamin C contents were measured as earlier described (Jacques-Silva et al. 2001).
Renal antioxidant status
The Superoxide dismutase (SOD) assay was carried out by the method of Misra and Fridovich (1972), with slight modification (Oyagbemi et al. 2015). The increase in absorbance at 480 nm was monitored every 30 s for 150 s. The one unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline to adrenochrome. Reduced glutathione (GSH) was estimated by the method of Jollow et al. (1974). Glutathione peroxidase (GPx) activity was also measured according to Beutler et al. (1963). Glutathione S-transferase (GST) was estimated by the method of Habig et al. (1974) using 1-chloro-2, 4-dinitrobenzene as substrate. The protein and non-protein thiol contents were determined as described by Ellman (1959).
Estimation of serum nitric oxide concentration and total protein
The serum nitric oxide concentrations were measured spectrophotometrically at 548 nm as previously described (Olaleye et al. 2007). Protein concentration was determined by the Biuret method of Gornal et al. (1949), using bovine serum albumin (BSA) as standard.
Small pieces of kidney were fixed in 10% formalin, embedded in paraffin wax, and sections of 5-6 mm in thickness were made and thereafter stained with hematoxylin and eosin (H&E) for histopathological examination according to the methods as previously described (Drury et al. 1976). Thereafter, the sections were examined with light microscopy.
Immunohistochemistry was done as described by Oyagbemi et al. (2019). Antibodies against renal angiotensin converting enzyme (ACE) and mineralocorticoid receptor (MCR) were probed in the heart using 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System with DAB solution (Catalog number: E-IR-R217 from Elabscience Biotechnology®, China). The kidney samples were fixed with 10% paraformaldehyde, embedded in paraffin and sectioned at a thickness of 5 μm. The slides were subsequently dewaxed in xylene (100%) solution for 2 minutes and afterward, hydration was carried out in different concentrations of ethanol (100%, 90%, and 80%) for 2 minutes each. The hydrated slides were rinsed and put in a PBS buffer tank for 5 mins. The antigen retrieval was performed with citrate buffer solution containing 2.1 g of citric acid monohydrate and 14.75 g of trisodium citrate dehydrate adjusted to pH 6.0 in microwave oven. Endogenous peroxide (H2O2 block) was carried out following manufacturer’s instructions as directed on the kit (E-IR-217C). Drops of H2O2 were added to cover the sections and incubated in humidifying chamber at room temperature for 10 min. The slides were rinsed afterwards and put back in the PBS tank for 5 min. Goat serum (E-1R-R217A) was added onto the slides to prevent nonspecific binding and incubated in humidifying chamber at room temperature for 30 mins. After 30 mins of incubation, the tissues were probed with primary antibodies viz-a-viz Angiotensin Converting Enzyme1 polyclonal antibody (E-AB-16159: 1:500 Dilution) and anti-mineralocorticoid receptor polyclonal antibody (E-AB-70261: 1:500 Dilution), and incubated for 2 hours at room temperature. Following incubation, the slides were rinsed with PBS and secondary antibody labelled (E-1R-R217B) was added, and the slides were incubated in humidifying chamber at room temperature for 20 min. Thereafter, the slides were rinsed and immersed in PBS tank for 5 min. Finally, a few drops of the substrate diaminobenzidine (DAB) was added at room temperature for 10 s; 50 µL of DAB concentrate (E-1R-R217D) + 1 mL DAB solution (E-1R-R217E) in the dark. The reaction was terminated with deionized water and slides were immersed in hematoxylin (Sigma-Aldrich, USA) for 3 s before rinsing with PBS. The slides were placed in 80%, 90%, and 100% of ethanol, and then xylene (100%) for 2 minutes each. Slides were removed, allowed to dry and a DPX mountant was applied. Sections were observed with a light microscope (Leica LAS-EZ®) using Leica software application suite version 3.4 equipped with a digital camera.
Data obtained were analyzed with One-way ANOVA with Dunnett’s post-test at a 95% confidence limit. All values are expressed as mean ±S.D. The test of significance between two groups was estimated by Student’s t test.