Ethics statement

The procedures conducted in this study received approval from the 'Research Ethics Committee of the Experimental Animals Use and Care', Faculty of Pharmacy, Beni-Suef University (BSU-IACUC, Egypt); Approval number 022–352 (with additional approval obtained from Vet, Cairo University, IACUC, Egypt; Vet CU 03162023635). The guidelines of the 'Animal House Rules follow the conventional guidelines of National Institutes of Health (NIH).

Animals

We got adult male Wistar rats weighing 150–180 g were obtained from the National Research Center, Cairo, Egypt. Animals were kept in plastic cages and maintained in an animal care facility under standard conditions of controlled temperature (25 ± 1 °C), humidity (50% ± 10%), and 12-h light/dark cycles. They were allowed for free access to standard food and water ad libitum, and were left for one week to acclimatize before any experimental procedures.

DrugsMethotrexate (MTX)

MTX was brought from Baxter Company (Cairo, Egypt) and was given to the rats by single i.p. injection (in a dose of 20 mg/kg at the 5th day).

Saponin from Quillaja saponaria Molina

Quillaja bark saponin (QBS) powder (S4521) was obtained from Sigma-Aldrich (St. Louis, MO), dissolved in normal saline and given orally to the animals (in a dose of 100 mg/kg/day, for 10 days) [3]. The major aglycone (sapogenin) that is mostly found in QBS is the triterpenoid; quillaic acid (pentacyclic triterpene). It is a hydroxy monocarboxylic acid and an aldehyde consisting mainly of 30-carbon atoms of the Δ12-oleanane type (C30H46O5), that was characterized by NMR spectroscopy, mass spectrometry and chemical methods) [24]. The methods for isolation, quantification and quality control of the purified saponin are reported in Sigma Product information Data Sheet (S4521).

Chemicals and kits

N-(1-naphthyl) ethylenediaminedihydrochloride (NEDD), Ellman’s reagent, MDA, reduced GSH and thiobarbituric acid, were obtained from Sigma-Aldrich (St. Louis, MO). The 3,3’-diaminobenzidine tetrahydrochloride (DAB) was acquired from Vector Laboratories Inc, located in Burlingame, California, USA. Rabbit polyclonal; Bcl-2 antibody (ab194583) and cleaved caspase-3 antibody (NB100-56113) were purchased from Abcam Biochemicals and Novus Biologicals, respectively. GF-1 Total RNA extraction Kit was obtained from Vivantis Technologies (Sdn Bhd, Malaysia). cDNA synthesis Kit was obtained from Bio-Rad, CA, USA. All other chemicals used were of the highest standard analytical grade.

Experimental design

A total of thirty-two rats were categorized into four groups (N = 8 each). Group I (Control group): represents rats that orally got 0.9% normal saline (vehicle of saponin; 0.2 ml/day, for ten consecutive days), in addition to receiving a single i.p. injection of 0.9% saline (vehicle of MTX) on day-five only. Group II (saponin alone-treated group, QBS): includes rats that received oral QBS (100 mg/kg/day) dissolved in normal saline (for ten consecutive days), in addition to a single i.p. injection of 0.9% saline on day-five only. Group III (MTX): contains the rats that received oral 0.9% normal saline (for ten days), along with a single i.p. injection of MTX (20 mg/kg) on the 5th day of the trial. Group IV (MTX + QBS): comprises the rats that orally administered the saponin (100 mg/kg/day, p.o, for ten days) and a single i.p. dose of MTX (20 mg/kg) at the 5th day of the experiment; 2 h prior to saponin administration. Twenty-four h after the last saponin dose, animals were anesthetized with i.p. thiopental sodium (75 mg/kg) and the blood samples were obtained using heparinized microcapillary tubes from retroorbital plexus, and processed for biomarkers assessments, as previously described [3]. After collection of blood, rats were sacrificed by cervical dislocation and the kidneys were immediately removed and washed three times using ice-cooled normal saline. The right kidneys were fixed in 10% phosphate buffered formalin for histological assessments and immunohistochemical examinations of Bcl-2 and cleaved caspase-3 [25]. The left kidney in each group was divided into two parts; one part was homogenized (1/10 w/v) in ice-cold Tris–HCl buffer (0.1 M, pH 7.4) for the preparation of 10% tissue homogenate and kept in − 20 °C for biochemical assays (determination of oxidative stress and inflammatory markers) [26]. The second part was snap-frozen in liquid nitrogen and kept in − 80 °C for qRT-PCR studies (3 animals/group) for determination of Nrf-2and Keap-1 mRNA expression levels [27].

Biochemical assaysKidney function analysis

Serum non-protein-nitrogenous substances; BUN (IFU/UREFSR01/00) and creatinine (IFU/CREFSR03/01), were used as biomarkers for kidney function and were estimated spectrophotometrically (UV-1700 Spectrophotometer, Shimadzu, Japan) using commercial kits (Meril life diagnostic kit, Gujarat, India) according to manufacturer's instructions [28].

Renal oxidative stress assessment

Kidney homogenate (10%) was obtained by adding 1 g of the renal tissue with 9 volumes of ice-cooled phosphate buffered saline (PBS) using IKA homogenizer (Model T 25 ULTRA-TURRAX, Staufen, Germany). The resultant homogenates were centrifuged (at 3000 × g), and the attained supernatants were processed using standard methods for determination of renal; GSH, as an index for renal antioxidant activity [29], MDA, as an index of the extent of lipid peroxidation in the kidneys [30], and NO content, as an indicator for oxidant free radicals in renal tissues [31].

Histopathological and immunohistochemical investigationsHistological analysis

Normal saline was used to wash the tissue samples of the kidneys that were fixed in 10% phosphate buffered formalin for 72 h in tightly-sealed containers. Samples were routinely processed in serial grades of ethanol, cleared in Xylene, and then impregnated into paraplast tissue embedding media (Leica Biosystems). Paraplast-embedded tissue blocks were cut at 4 μm thickness using rotatory microtome then placed on glass slides and stained with hematoxylin and eosin (H&E) [32]. Tissue sections were investigated under light microscope for demonstration of common indices of kidney histology in different samples, and representative images were shown.

Evaluation of apoptosis by immunohistochemical determination of Bcl-2 & cleaved caspase-3

Preparation for immunohistochemical staining, including sections deparaffinization, and antigens retrieving was conducted. After blocking non-specific protein binding, sections were washed by PBS and incubated overnight (at 4 °C) with the primary rabbit antibody against rat; cleaved caspase-3 (NB100-56113) from Novus Biologicals, (dilution 1:1000), or Bcl-2 (ab194583) from Abcam Co (Cambridge, MA, USA), (dilution 1:100). Then, sections were washed by PBS, incubated with secondary antibody; HRP Envision kit (DAKO), for 20 min, washed by PBS, and positive immunoreactivities were developed by DAB visualization for 10 min and counter-staining with hematoxylin. Quantitative analysis was performed according to El-Nabarawy et. al. (2020) for determination of area percentage of immunohistochemical expression levels of indicated proteins, as estimated from six representative randomly selected fields in the tissue section using Leica application software (Leica Microsystems GmbH, Wetzlar, Germany) [33]. Statistical analysis of renal immuno-expressions in different groups was carried out using chi-squared "χ2" test. Representative microscopic images (× 400) were shown in the study.

Evaluation of inflammationDetermination of renal mRNA levels of Nrf-2and Keap-1, using Real time PCR

Total RNA was isolated from renal tissues using GF-1 Total RNA Extraction Kit (GF-TR-050, Vivantis Technologies Sdn Bhd, Malaysia), according to the manufacturer’s instruction. The isolated RNA was treated with a DNase I (RNase-free kit; Fermentas, MD, USA). After complementary DNA (cDNA) synthesis (Script™ cDNA synthesis kit; Bio-Rad, CA, USA), the quantitative real time-polymerase chain reaction (qRT-PCR), was conducted as previously described [3]. After PCR amplification, the ΔCt was calculated by subtraction of the β-actin Ct from each sample Ct., and relative levels of gene expression were determined. The β-actin is used as a reference gene. Table 1 shows the sequences of Nrf-2 and Keap-1 primers.

Table 1 The sequences of the oligonucleotide primers used for RT-PCREstimation of renal TNF-α

Along with the modulations of Nrf-2/Keap-1 mRNA levels, the protein level of renal TNF-α was used as a marker for inflammation in the current study. The level of TNF-α in the homogenates of the kidneys tissues were quantitated using ELISA assay (MBS175904), according to the manufacturer’s instructions (My BioSource, St. Louis, MO, USA) [34].

Statistical analysis

Values were reported in the form of the means ± SEM. The various treatments were compared by the One-way Analysis Of Variance (ANOVA) test followed by Tukey–Kramer comparisons test applied across the four groups or Chi-Squared "χ2" test wherever indicated. The results were deemed to be significantly different at p < 0.05. Data analysis was accomplished using the computer software GraphPad prism, SanDiego, USA.