Co-treatment with esculin and erythropoietin protects against renal ischemia-reperfusion injury via inhibition of P2X7 receptors and activation of PI3K/Akt

plant material

Vachellia farnesiana flowers were collected from Al-Qaliobia Governorate, Egypt in March 2020. Identification and authentication of the collected plant was carried out by Dr. Terase Labib, Department of Flora and Taxonomy, El-Orman Botanical Garden, Giza, Egypt. A reference specimen (n° Vf/fl/2020) is kept in the garden herbarium. Experimental research and field studies on plants, including the collection of plant material, were approved by the IRBT Research Ethics Board and complied with relevant institutional, national and international guidelines and legislation.

Extraction and splitting

fresh flowers from V. farnesiana (1.350 kg) were soaked for 3 days in 85% methanol (5 × 2 l) at room temperature. The combined extracts were filtered and evaporated in vacuo using a rotary evaporator to obtain an 85% methanol extract (132.85g). The dried methanol aqueous extract was defatted using petroleum ether (60–80°C). Then, the defatted methanol extract underwent successive fractionation using organic solvents (e.g., dichloromethane, ethyl acetate and not-butanol) in order to obtain 17.36, 15.85, 21.43, 43.74 and 31.18 g for petroleum ether, dichloromethane, ethyl acetate, not-butanol and aqueous extracts, respectively.

Chromatographic isolation and purification of the ESC compound

The chemical constituents of the tested extract were tentatively identified using a Thermo Finnigan (Thermo electron Corporation, OK, USA), coupled to an LCQ Duo ion trap mass spectrometer with an ESI source in dimmer mode. negative ionization (ThermoQuest Corporation, Austin, TX, USA)⁵⁴. The ethyl acetate extract (20.0 g) was then fractionated via column chromatography (5 × 60 cm) packed with polyamide 6S as stationary phase and eluted via a mixture elution system. gradient (water: MeOH). A total of five major subfractions (F1-F5) were obtained. The obtained subfractions (F1-F4) were separately subjected to further further purification using several Sephadex LH-20 subcolumns (2 × 30 cm) eluted with (water:MeOH with MeOH gradient) to get gallic acid, methyl gallate, p-coumaric acid, quercetin, taxifolin, naringenin and quercetin 3-O-glucoside. While F5 (5.2 g) eluted with 85% MeOH from the main polyamide column was subjected to several Sephadex LH-20 sub-columns for further purification eluted with methanol:water in an elution system at gradient to get the ESC.

Animals

Forty male Sprague-Dawley rats weighing 280-300 g were kept in the animal facility of Theodor Bilharz Research Institute (TBRI), Egypt. Rats were maintained in individual polypropylene cages at 22–25°C, with a 12–12 h dark-light cycle and free access to standard laboratory diet and water. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Eighth Edition) of the National Institutes of Health, consistent with ARRIVE guidelines, and approved by the TBRI Research Ethics Board for conduct animal experiments ( PT: 584; 2/22/2021).

Surgical interventions

Each rat was anesthetized with 6% Desflurane (Desflurane®, Baxter, UK) in 100% v/v oxygen by inhalation. The rats were placed in the supine position, the abdominal skin was shaved after disinfection with 70% alcohol and a midline laparotomy incision was made. Subsequently, both kidneys with their pedicles were gently exposed and non-traumatic vascular clamps (Bulldog clamps) were placed bilaterally over both the artery and the vein of each kidney. After 45 min, successful reperfusion was achieved by removing the clamps, restoring blood supply. The surgical incisions were stitched up and covered with sterile gauze. After recovery from anesthesia, the rats were returned to their sterile cages for 24 h with free access to food and water. An identical operation was performed for the sham group without clamping the bilateral renal pedicles.

Experimental design

The rats were randomly divided into five groups of eight rats in each group. The groups of animals were as follows; Sham (vehicle received alone), vehicle + RI/R, ESC + RI/R, EPO + RI/R and ESC + EPO + RI/R. Rats were pretreated with ESC (50 mg/kg¹¹) or vehicle (0.5% carboxymethylcellulose in phosphate buffered saline (PBS), Sigma Aldrich, USA) by oral gavage for 7 days and 30 min before RI/R. Meanwhile, EPO (Eprex®, Janssen-Cialg, Switzerland) was administered in a single dose of 1000 U/kg, IP, 30 min²⁰ before IR/R. Twenty-four hours after reperfusion, rats were sacrificed under light anesthesia by inhalation of isoflurane (Forane®, Baxter, UK), then blood and kidney tissues were collected and processed for biochemical examinations, RT – Subsequent PCR, histopathology and immunohistochemistry (IHC). .

Biochemical analyzes

Collected blood samples were centrifuged at 3000×g for 10 min, then sera were separated and stored at -80°C for spectrophotometric evaluation of creatinine and urea levels (Biodiagnostics, Egypt), while levels of neutrophil gelatinase-associated lipocalin (NGAL ) were determined using the commercial enzyme linked immunosorbent assay (ELISA) kit (MyBiosource San Diego, USA), according to the manufacturer’s instructions.

Additionally, part of the right kidney was homogenized in ice-cold PBS and the supernatants were used for measurement of malondialdehyde (MDA), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels using specific assay kits, as described by the manufacturer. (Biodiagnostics, Egypt). Additionally, levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, NF-κB p65, endothelial nitric oxide synthase (eNOS), p-PI3K, p-Akt , bax and bcl2 were quantified using ELISA kits according to the manufacturer’s instructions (MyBiosource San Diego, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR).

Total RNA was isolated from kidney tissues homogenized with Easy-spin RNA extraction kit (Intron biotechnology, Korea). The RNA was then converted into its complementary DNA (cDNA) RevertAid First Strand cDNA Synthesis Kit (Thermoscientific, USA). qRT-PCR was performed with StepOne™ Real-Time PCR (Applied Biosystems, USA) using Maxima SYBR Green qPCR Master Mix, no ROX (2×) (Thermoscientific, USA), according to the manufacturer’s instructions. Reverse transcription reaction conditions were run at 42°C for 15 min, followed by 3 min at 95°C. Thermal cycling conditions were run at 95°C before denaturation, followed by 40 cycles (95° C for 15 s; 60°C for 30 s; 72°C for 60 s). The following oligonucleotide primers were used: 5′-GTGGAGATCCTAGGTTTCTCTG-3′ (forward), 5′-CAGGATCTCATTCTCTTGGATC-3′ (reverse) for NLRP3, 5′-TTTCTTCCCCTACATCCTGCT-3′ (forward), 5′-CTGTCAGAAGTCTTGTGCTCTG-3′ (antisense) for Caspase-1, 5′-CTACTCTTCGGTGGGGGCTT-3′ (sense), 5′-CTCTGGATCCGGGTGACTTT-3′ (antisense) for P2X7R, 5′-ACACCACGGTTTGGACTATGG-3′ (sense), 5′-GGCTACAGTAGTGGGCTTGG-3′ (antisense) for PI3K, 5′-ATGTCCGAGATCCTACCCTACG-3′ (sense), 5′-AGCGAAGAAGGAGTGGTGTC-3′ (antisense) for Akt and 5′-TGATACAAAGACGGGACATCG-3′ (sense) and 5′-CACGATTTCCCTCTCAGC-3′ (antisense ) for β-actin as an internal control for normalization of target genes according to 2^−ΔΔCt method⁵⁵. All qRT-PCR expression experiments were performed in triplicate to ensure reproducibility of the results obtained.

Histological examination

The kidneys were grossly examined for corticomedullary demarcation, hemorrhage, and necrosis. Next, the left kidney samples were divided into two parts, one for histopathology and the other for electron microscopy examinations. Briefly, kidney tissues were immediately fixed in 10% formalin solution, processed, and embedded in paraffin. Sections 4 microns thick were then stained with: (1) hematoxylin and eosin (H&E) staining for histological evaluation of parenchymal injury (necrosis of the tubular lesion, interstitial inflammatory infiltrate and glomerular changes), (2) Periodic Acid Schiff (PAS) reagent to demonstrate capillary and tubular basement membranes, and (3) Masson’s Trichrome (MTC) stain to assess fibrosis and demonstrate Fibrinoid necrosis and fibrinous thrombosis At least 10 cortico-medullary fields were examined in each section at different magnifications, and a semi-quantitative analysis of the tubulointerstitial lesion was performed. Total tubular injury was scored on a scale of 0 to 4 based on percentage of normal tubules and extent of injury as follows: 0, absent; 1 (minimal, 6-10%)); 2 (Focal, 11–25%)); 3 (Moderate, 26–50%)); 4 (Diffuse, >50%).

Transmission electron microscope (TEM)

Kidney samples for TEM were fixed in 2.5% glutaraldehyde in cacodylate buffer for 2 h at 4°C. Tissues were then washed twice for 1 h each in cacodylate-sucrose buffer and post-fixed for 1 h at 4°C in 2% osmium tetroxide. After dehydration in ascending graded ethanol, the samples were impregnated with Epon 812 substitute (EMBed-812 kit, Electron Microscopy Science, USA) at room temperature and polymerized at 60°C for 48 h. Semi-thin sections were cut, stained with methylene-azure blue II and examined under a light microscope to choose the region of interest for the ultra-thin section. Ultrathin sections at 70 Å thick were then prepared using an Ultracut R ultramicrotome (Leica, Vienna, Austria) and stained twice with uranyl acetate and lead citrate. Ultrastructural examinations of glomerular capillary loops, basement membrane, podocytes and signs of tubular damage were performed at 80 kV with the Philips EM 208 S electron microscope (Philips Optics, Eindhoven, The Netherlands) provided by the Department of electron microscopy of the TBRI.

Immunohistochemical examinations of caspase-3 activity

Sections (5 microns thick) of formalin-fixed, paraffin-embedded tissue specimens were prepared on loaded and deparaffinized glass slides, hydrated, and then treated for antigen retrieval at elevated pH (pH = 8) using an automated immunostainer (Sigma, Aldrich, USA). Rabbit polyclonal anti-caspase antibodies (Sigma, Aldrich, USA); at a dilution of 1:200 was used to detect apoptosis. Biotinylated goat anti-rabbit/HRP immunoglobulins (Sigma, Aldrich, USA) were used at a dilution of 1:300. The detection method for streptavidin-biotin-peroxidase complex and peroxidase-DAB (3,3′-diaminobenzidine) (Sigma, Aldrich, USA) was carried out according to the manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin. Positive and negative control slides were included in each run. As a negative control, a tissue section was treated as described but with the primary antibody omitted.

statistical analyzes

All data are expressed as mean ± SD. Statistical analyzes were conducted using one-way analysis of variance (ANOVA) followed by post hoc Tukey–Kramer multiple comparison test for parametric analysis, while Kruskal–Wallis test followed by the Dunn’s post hoc was used for the nonparametric analysis. Statistical differences were assessed using GraphPad Prism software (USA, version 5.03). Statistical significance was set at ppp